Dental caries is a multi-factorial oral disease, requiring a susceptible host, cariogenic microorganisms and suitable substrate. Caries is extended worldwide in spite of the availability of countless prophylactic means, including fluoride toothpaste and dental sealers. Many efforts have been made to achieve isolation of pure natural products for medicinal use. Flavonoids are bioactive polyphenol compounds possessing multidimensional effects such as antibacterial action.Agreggatibacter actinomycetemcomitans, Actinomyces naeslundii, Actinomyces viscosus, Enterococcus faecalis, Escherichia coli, Lactobacillus casei, Staphylococcus aureus, Streptococcus oralis and Streptococcus sanguinis as well as Candida albicans fungal strain.The present study targeted the characterization of antibacterial and antifungal activity of various flavonoids . Nine strains present in dental plaque were used: Results revealed that luteolin, morin, naringin, quercetin and rutin effectively inhibited bacterial and fungal growth. However, morin was the most effective flavonoid.It might then be concluded that flavonoids show bacteriostatic effect on all of tested bacteria and fungus. Biochemistry; Microbiology; Pharmaceutical science; Pharmacology; Flavonoids; Dental plaque; Caries However, it has been isolated in suppurative parotitis, angular chelitis and dentoalveolar infections [Enterococcus faecalis is a Gram-positive facultative coccus that may opportunistically colonize periodontal pockets and contribute to periodontal breakdown in heavily infected sites [Dental plaque is a microbial community that colonizes the surfaces of the teeth in a structurally and organized biofilm , 2. Micrfter 8 h , 8, 9. Abacillus . Prevalefections . Enteroced sites .Candida albicans has been shown to be the predominant pathogen; it is an innocuous commensal of microbial communities in human oral cavity [Candida becomes virulent it generates candidiasis, which can be manifested through various clinical forms, involving one or more oral sites followed by total mouth affectation [Microorganisms present in the oral cavity interact using their metabolic products or exchanging molecular signals. l cavity . Its pril cavity . When Caectation .Streptococcus colonization by focusing on removing the whole dental plaque [In order to reduce dental caries, numerous studies have been conducted. Dental therapy is primarily targeted through the prevention of l plaque ; this prl plaque . Some otl plaque . Ecologil plaque .Escherichia coli, Staphylococcus aureus, Streptococcus mutans, Streptococcus sanguinis and Actinomyces viscosus [Flavonoids, a family of polyphenolic compounds, widely distributed in the plant kingdom are consumed in significant amounts as part of the human diet . Differeviscosus , 20, 21.Streptococcus mutans within biofilms [Porphyromonas gingivalis with MIC values in the range of 62.5–125 μg/mL [Salmonella enteritidis, Escherichia coli, Bacillus subtilis and Staphylococcus aureus [Candida albicans to prevent Candidiasis.Recently, some research groups have identified two potential anti-caries agents found in propolis, a natural bioactive product: apigenin  a non-toxic dietary flavonoid found in fruits and vegetables. It displays activity against streptococcal membranes by increasing their proton permeability as well as inhibiting acid production by biofilms , 23. Lutbiofilms . Myricets aureus , 27. Difs aureus . Since p22.1Apigenin, catechin, morin, myricetin, luteolin, naringin, quercetin and rutin all within a purity range of <95% were obtained from Sigma-Aldrich . The structures of the flavonoids used in this study are shown in 2.2Candida albicans  was grown in Sabouraud agar  for 24 h at 37 °C. Identity was confirmed by conventional identification methods, such as germ tube induction at 37 °C, microscopic morphology and chlamydospore formation in corn meal agar . Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 15987) were grown in Tryptic Soy Agar broth containing 1 μg/mL of yeast extract, 5 μg/mL of hemin and 1 μg/mL of menadione in an anaerobic system in atmosphere of 80% N2, 10% CO2 and 10% H2 at 35 °C for 16 h. Blood Agar  adding 5 mL of hemin (0.05% concentration) and 5 mL of Vitamin K stock (0.005% concentration) at anaerobic conditions and 37 °C for 48 h. Lactobacillus casei (ATCC 393) was grown in Man, Rogosa and Sharpe (MRS) broth agar  and incubated at 37 °C for 16 h. Enterococcus faecalis(ATCC 8043) was grown in Trypticase Soy Broth , 1 μg/mL yeast extract and 15 g/L agar  and incubated at 37 °C in a reduced O2 atmosphere. Blood Agar  adding 5 mL of hemin (0.05% concentration) and 5 mL of Vitamin K stock (0.005% concentration) at anaerobic conditions and 37 °C for 24 h. Escherichia coli (ATCC 25922) was grown in Luria Bertani Miller broth containing 1% NaCl, 1% peptone and 0.5% yeast extract. Streptococcus sanguinis (ATCC 10556) and Streptococcus oralis (ATCC 35037) were grown in Trypticase Soy Agar broth with defibrinated sheep blood at 37 °C in 5% CO2 atmosphere. Blood Agar  adding 5 mL of hemin (0.05% concentration) and 5 mL of Vitamin K stock (0.005% concentration) at anaerobic conditions and 37 °C for 48 h. Agreggatibacter actinomycetemcomitans (ATCC 43718) was grown in Blood Agar  adding 5 mL of hemin (0.05% concentration) and 5 mL of Vitamin K stock (0.005% concentration) at anaerobic conditions and 37 °C for 48 h. Staphylococcus aureus (ATCC 25923) was grown in Blood Agar  adding 5 mL of hemin (0.05% concentration) and 5 mL of Vitamin K stock (0.005% concentration) at 37 °C.All strains were stored at -80 °C in the medium with 20% glycerol (v/v). Prior to use, the aliquots of these cultures were reactivated in their medium incubated at 37 °C for 24 h. All mediums were prepared according Manufacturer's instructions.2.32SO4) was added to 99 mL of distilled water and mixed thoroughly. After this, 1% v/v of barium chloride (BaCl2) solution was achieved by mixing 1.0 g in 100 mL of distilled water. Concentrated H2SO4 solution was mixed with the barium chloride solution to produce a turbid solution of barium sulfate (BaSO4). After that, a small volume of the turbid solution was transferred into a capped tube, of the same type as that used for test and control inocula, after this it was stored in a well-sealed container at temperatures between 20-28 °C [One mL of concentrated sulfuric acid  (SIGMA) dissolved in dimethylsulfoxide, which was used as a vehicle. And Ampiciline 0.1 μg/mL as a positive control.2.68 CFU/mL) was used to inoculate Blood Agar + Hemin + Vitamin K, Sabouraud, LB and BHI (according to the bacteria/fungi) plates evenly using a sterile swab. Flavonoid-impregnated discs were individually placed on the Blood Agar, Sabouraud, LB or BHI surface with flamed forceps and gently pressed down to ensure contact with the agar surface. Discs were impregnated with flavonoids, at increasing doses between 0.1 mg/mL and 1.0 mg/mL every 0.1 units and 5.0 mg/mL. Five discs per plate were placed in order to avoid both reflection waves from the edges of the petri dishes and overlapping inhibition rings. The plates were then incubated at 37 °C for around 18 h in inverted position to look for zones of inhibition. And some in anaerobic conditions. As soon as we observed growth inhibition of bacteria, the stocks were diluted in order to get exact doses of flavonoid.Disc diffusion method for antimicrobial susceptibility test was carried out according to the standard method designed by Kirby-Bauer to evaluate the presence of antimicrobial activities of flavonoids. A bacterial suspension adjusted to 0.5 McFarland standard . Luteolin only had impact at the 5 mg/mL (1.7 × 10−2M) concentration for A. viscosus while Morin at 10 mg/mL (3.3 × 10−2 M) for both strains. Also, Naringin had an impact only on A. naeslundii at 10 mg/mL concentration (1.7 × 10−2 M) and 30 mg/mL (5.1 × 10−2M). Finally, quercetin showed an activity at 10 mg/mL (3.3 × 10−2 M) and 30 mg/mL (9.9 × 10−2M) for both strains. In the case of bA.n. there are significant differences between rutin, quercetin, naringin and morin (ρ < 0.05), and for cA.v. there are significant differences between luteolin, morin and rutin (ρ < 0.05).We found that rutin, quercetin and morin showed antibacterial activity against Actinomyces, and luteolin for A. naeslundii .Apigenin, rutin, luteolin, naringin and morin showed some activities against acterium . For eE.3.3Escherichia coli growth. We found that rutin, luteolin and naringin at 100 μM doses inhibited the growth of Gram-negative bacteria strain, Escherichia coli. Rutin exhibited inhibition to a higher concentration of 1 mM. It was also found that at higher concentrations they also showed inhibitions from naringin at 30 mg/mL (5.1 × 10−2 M). Morin 10 mg/mL (3.3 × 10−2 M) and quercetin at 10 mg/mL (3.3 × 10−2 M)  .3.4Lactobacillus casei. Rutin and naringin each showed antibacterial activity at 100 μM, but morin was active at higher doses of 1 mM (fL.c. we can observe significant differences between naringin and rutin (ρ < 0.05).The antibacterial activity was analyzed through paper disk method using  of 1 mM . For fL.3.5Staphylococcus aureus. Bacteria exhibited sensitivity to apigenin, luteolin, morin and rutin at doses of 1 mM. Bacteriostatic effect was shown from quercetin at 0.05 mg/mL (1.6 × 10−4 M), 0.01 mg/mL (3.3 × 10−5 M), 0.5 mg/mL (1.6 × 10−3 M), 0.1 mg/mL (3.3 × 10−4 M), 1 mg/mL (3.3 × 10−3 M) and 10 mg/mL (3.3 × 10−2 M) and 30 mg/mL (9.9 × 10−2 M). Luteolin 1 mg/mL (3.4 × 10−3 M) and 5 mg/mL (1.7 × 10−2 M). Morin 1 mg/mL (3.3 × 10−3 M) and 10 mg/mL (3.3 × 10−2 M). Rutin 1 mg/mL (1.6 × 10−3M), catechin 1 mg/mL (3.4 × 10−3M) and 10 mg/mL (3.4 × 10−2 M), naringin 1 mg/mL (1.7 × 10−3 M), 10 mg/mL (1.7 × 10−2 M) and 30 mg/mL (5.1 × 10−2 M) (gS.a. we found significant differences between rutin and luteolin with catechin and apigenin (ρ < 0.05).The antimicrobial activity of the flavonoids was assessed on  10−2 M) . For gS.3.6−2 M), quercetin 1 mg/mL (3.3 × 10−3 M),10 mg/mL (3.3 × 10−2 M), 30 mg/mL (9.9 × 10−2 M), catechin 10 mg/mL (3.4 × 10−2 M), luteolin 1 mg/mL (3.4 × 10−3 M), luteolin 5 mg/mL (1.7 × 10−2 M) and naringin 30 mg/mL (5.1 × 10−2 M) .Effect from flavonoids against the anaerobic bacteria was shown from morin 10 mg/mL (3.3 × 10 10−2 M) . For aA.3.7S.o. and S.s. there is no significant difference between all of the flavonoids tested.There was not shown any antibacterial effect to these bacteria strains . For S.o3.8−2 M) from Quercetin and Morin was shown to inhibit/stop Candida albicans growth. Luteolin inhibit C. albicans at 5 mg/mL (1.7 × 10−2 M) .Rutin and naringin exhibited inhibitory effect at doses of 100 μM; higher doses were required for morin (1 mM) to show inhibitory effect. An effect at 10 mg/mL (3.3 × 10 10−2 M) . For dC.4Actinomyces naeslundii, Actinomyces viscosus, Aggregatibacter actinomycecomitans, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus and Lactobacillus casei while apigenin showed activities against Enterococcus faecalis and Staphylococcus aureus. Catechin only had antibacterial activity against A. actinomycecomitans and S. aureus. We also found that luteolin, naringin, morin, quercetin and rutin inhibited Candida albicans. Morin had the most prevalent inhibition against 7 different strains of bacteria and C. albicans.Data showed that luteolin, morin, naringin, quercetin and rutin inhibited Actinomyces, Escherichia coli, Staphylococcus aureus and Enterococcus faecalis are known as multidrug resistant organisms and are a particular hazard to medical practice [Streptococcus mutans and Streptococcus sobrinus [Streptococcus to adhere to hydroxyapatite pellets pretreated with saliva showed that flavonoids significantly inhibited adhesion to the pellets [Streptococcus mutans within biofilms [Streptococcus mutans is not the only organism causing caries. Dental caries etiology clearly points out its multi-factorial nature. Many other microorganisms with time, dietary factors and host factors promote caries. In the present study, we found that flavonoids exert activity against Gram-positive and Gram-negative microorganisms and against Candida. Recently, it has been shown that procyanidins disrupted acid production and acid tolerance of Streptococcus mutans. Further studies using individual cariogenic microorganisms shall elucidate the biological effects of flavonoids, they will advance our understanding on exact action mechanisms of these compounds. The susceptibility of certain strains of bacteria towards test compounds was evaluated by measuring inhibition diameter, from here it follows that increase of flavonoid concentration does not have any substantial influence on their antibacterial activity, this could be the result of a poor diffusion of these flavonoids in the agar plates.Pathogenic strains of species such as practice , 31. Presobrinus , 33. Res pellets . Furtherbiofilms , 37, 38.Staphylococcus growth.However, molecule structure is closely related with their biological activity. Flavonoids tested in this study belong to polyhydroflavones. The molecules contain hydroxyl group substitutions at R3, R7 and R4’. Myricetin differs from the rest of flavonoids with a hydroxyl substitution on R5′, which represents just one hydroxyl group modification therefore has no antibacterial activity. Some researchers suggested that 2′, 4′-dihydroxylation of B ring and 5,7-dihydroxylation of the A ring are important for a significant inhibition of In conclusion results presented in this research provide evidence that naturally occurring flavonoids exhibit antibacterial activity. Further research need to be conducted in order to establish the role of flavonoids in dental plaque control.Gloria Gutiérrez-Venegas: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Juan Arturo Gómez-Mora: Conceived and designed the experiments; Performed the experiments.Marco Antonio Meraz-Rodríguez, Mónica Arisbet Flores-Sánchez: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.Laura Fabiola Ortiz-Miranda: Performed the experiments.Dirección General de Asuntos del Personal Académico PAPIIT-201816.The work was supported by grant from The authors declare no conflict of interest.No additional information is available for this paper.


These gold compounds were characterized by multinuclear NMR, microanalysis, mass spectrometry, and X-ray crystallography. The inherent electrochemical properties of the gold complexes were also studied by cyclic voltammetry and theoretical insight of the complexes was gained by density functional theory and TD-DFT calculations. The complexes effectively kill cancer cells with IC50 in the range of ~0.10–2.53 μΜ across K562, H460, and OVCAR8 cell lines. In addition, the retinal pigment epithelial cell line, RPE-Neo was used as a healthy cell line for comparison. Differential cellular uptake in cancer cells was observed for the compounds by measuring the intracellular accumulation of gold using ICP-OES. Furthermore, the compounds trigger early – late stage apoptosis through potential disruption of redox homeostasis. Complexes 1 and 3 induce predominant G1 cell cycle arrest. Results presented in this report suggest that stable gold-phosphine complexes with variable oxidation states hold promise in anticancer drug discovery and need further development.The reaction of gold reagents [HAuCl Exploring the Au(I) and Au(III) chemical space has given rise to enormous diversity of gold compounds of biological relevance, influenced by creative ligand design16. Despite effective clinical and preclinical treatment of cancer and rheumatoid arthritis by gold complexes such as auranofin, the molecular basis of drug action remains unclear for gold(III) phosphine compounds present in this report. Years of research implicates a number of disease targets including: (i) proteasome-associated deubiquitinases9; (ii) thiol-rich enzymes such as thioredoxin and glutathione reductase20; (iii) thiol-dependent proteases21; iv) autophagy induction22; and superoxide/oxyradical ion generation23.Gold-based probe development and drug discovery remain a burgeoning area of biological research and treatment for disease indications such as cancer25 and ovarian malignancies28 as well as microbial infections31 is a phosphinogold complex. This has accelerated the development and discovery of several gold-phosphine complexes for therapeutic applications. Gold(I)-phosphine anti-cancer complexes have been identified to trigger apoptosis by targeting the mitochondria and inhibiting thioredoxin reductase34. Structural diversity of gold complexes bearing phosphine ligands have important implications for anticancer activity and probe development35. Work by Berners-Price et al. demonstrated the anticancer effect of gold-phosphine complexes and have also tried to improve the in vitro and in vivo efficacy of this class of compounds42. Gold complexes bearing dithiocarbamate45 and triorganophosphine ligands46 of the type [(R3P) Au(S2CNR2)] display anticancer activity across a panel of cancer cells including ovarian cancer cells47. Recently, Darkwa and co-workers synthesized dinuclear phosphinogold(I) complexes bearing varied phosphine ligands including triphenylphosphine, and diphenylphosphino-alkanes and dithiocarbamates of the type [Au2Cl2(dppe)] and evaluated their anticancer activity47. The complexes displayed broad spectrum of activity in a number of cancer cell lines. Additionally, the anticancer activity of phosphinogold(I) complexes bearing thioglucose ligands as in the case of auranofin show higher potency than their thiolate counterparts even in cisplatin resistant cells. For example, the P – Au – S structural motif is prevalent in a number of gold-phosphine complexes such as the lupinylsulfide (OmS) or sulfanylpropenoate (sppa)48 containing phosphinogold(I), [AuOmS)2(Ph2P(CH2)2PPh2] or[Au(PPh3)(sppa)], respectively and they exhibit good anticancer activity49. Improving the biological activity of gold-phosphine complexes require ligand tuning that expand diversity, lipophilicity, physiological stability, and high selective cytotoxicity in cancer cells over normal cells51.Auranofin, which is under clinical and preclinical investigation for the treatment of a variety of cancers including leukemia55. These ligands impart strong σ-donating character to the gold center for stability and offer the possibility of different ligands around the metal center, given its square-planar geometry56. Che and co-workers showed that dinuclear cyclometalated gold(III) phosphine, [(C^N^C)2Au2(μ-dppp)]CF3SO3)2 inhibit hepatocellular carcinoma in vivo by inducing ER stress57. There still remains the need to expand the structural diversity of gold-phosphine complexes by designing new gold(III)-phosphine complexes.Whereas a lot of work has been conducted with linear phosphinogold(I), its high oxidation state counterpart gold(III) needs further exploration. Recent advancement of cyclometalated gold(III) in anticancer development show promising resultsAnother important feature of ligands in the context of biological efficacy is chirality, since they possess the property to tune substrates to respective biological targets for improved target engagement that may be elusive for non-chiral ones. The use of chiral ligands in gold drug discovery remain largely unexplored. Incorporating chiral ligands into gold(I) or gold(III) complexes will expand the chemical space to further opportunities in medicinal inorganic chemistry.In this report, we synthesized gold(I) complexes bearing chiral or achiral phosphine ligands and in addition mononuclear (C^N)-cyclometalated gold(III) bearing chiral or achiral phosphine ligands. The complexes display potent cytotoxic activity in different cancer cell lines by triggering apoptosis through ROS induction. The study establishes the need for a broader scope of gold complexes for cancer therapy.+3 + 2e− → Au+1E° = 1.41 V and Au+1 + e− → Au(s)Eo = 1.69 V)58. This is due to relativistic effects. Often, strong σ-donating ligands such as phosphines and N-heterocyclic carbenes are used to improved stability of gold complexes against rapid reduction59. The polarizable nature of gold, owing to its loosely held electrons facilitates its coordination to soft Lewis bases such as phosphorus, sulfur containing ligands. Linear gold(I) complexes are limited by their low coordination number. On the other hand, gold(III) compounds largely exist in a square-planar configuration and provides increased number of coordination sites. This can be harnessed for the development of anticancer agents bearing multidentate ligands with enhanced stability and activity. In addition the use of chiral phosphine ligands in gold scaffolds and their effects on geometry remains underdeveloped. A study that examines the effect of oxidation states of compounds bearing phosphine ligands will be beneficial. We envisioned the investigation of gold(I)/(III)-phosphino complexes as potential anticancer agents to offer preliminary structure-activity insights of gold-based metallodrugs. These gold(I)/(III) complexes could interact with biological targets, such as proteins, in a similar fashion to reported gold-phosphino complexes but with optimal kinetic lability to avoid premature deactivation. Within this context, work that studied the anticancer potential of gold-phosphino complexes offered impetus that the complexes synthesized in our laboratory will be active against cancer cells in vitro. Furthermore, the distorted gold(I) with phosphine ligands as well as gold(III) complexes bearing cyclometalated and phosphine ligands could exhibit the needed stability for effective therapeutic response, even in drug-resistant cancer.Stabilizing the gold metal center for biological utility remains an important aspect of metallodrug discovery. Gold compounds possess high redox potential -(-)-2,3-bis(t-butylmethylphosphino) quinoxaline and achiral 1,2-bis(diphenylphosphino)ethane (DPPE) phosphine ligands due to their biocompatibility and moderate lipophilicity when present coordination complexes or as ancillary ligands in organometallic compounds. For further investigation into the geometry and oxidation states of these complexes and their effects on anticancer activity, we explored the synthesis of dinucleur gold(I) complexes and (C^N)-cyclometalated backbone for the formation of an organometallic gold(III), all bearing phosphine ligands used in this study. We hypothesized that the small set of compounds with rich diversity including, ligand type, stereochemistry, oxidation state, geometry and distortion, would possess interesting biological properties for gold-based drug discovery.60, which is well tolerated in mice was used for the synthesis of the respective Au(I) and Au(III) compounds, using tetrachloroauric acid trihydrate (HAuCl4·3H2O) or AuCl(tht), or -cyclometalated Au(III)Cl2 starting reagents, demonstrated in compounds 1, 2, and 3. To expand the diversity of gold-phosphine complexes, we synthesized compounds with the archetypical achiral phosphorus-donor ligand, DPPE, exemplified in 463, 566, according to previously reported protocols but with different anions, and the novel gold(III), 6. The synthetic approach for compound 1, was achieved by adding a chloroform solution of -(-)-2,3-bis(t-butylmethylphosphino) quinoxaline to a cold solution of AuCl(tht) in chloroform. This complex was dried and recrystallized from chloroform/ether to give a pale yellow neutral compound. The synthesis of 2 was carried out by refluxing an equimolar concentration of HAuCl4·3H2O and -(-)-2,3-bis(t-butylmethylphosphino) quinoxaline in chloroform for 1 h. After filtering through celite the reaction was purified by flash silica-gel chromatography to obtain 2 as a brick-red monocationic solid. We synthesized gold(III) analogs using the benzyolpyridine (C^N)-cyclometalated complex, 7, which was synthesized as previously reported67. The chiral -(-)-2,3-bis(t-butylmethylphosphino) quinoxaline) and achiral (DPPE) bisphosphine ligands were used in a ligand substitution reaction with 7 to give 3 and 6 respectively, after purification by silica-gel column chromatography. Phosphines are strong coordinating ligands which can readily displace the chlorine atoms. In the reaction that leads to complex 3, the formation of 2 in low yields is observed. This is as a result of reductive elimination promoted by the nucleophilic phosphine. It is a subject of intense investigation within our laboratory68. Complexes 4 and 5 bearing DPPE ligands were synthesized to investigate the effect of perchlorate counter anions on cellular activity. The complexes were purified by recrystallization from acetonitrile as white cationic solids.The gold-phosphine complexes under investigation in this study are depicted in Fig. 1H, 13C, and 31P{1H} NMR spectroscopy-(-)-2,3-bis(t-butylmethylphosphino) quinoxaline and DPPE have single resonances at −17 and −12 ppm respectively. Upon coordination with the gold center, the resonances shift downfield. For the gold(III) complexes, 3 and 6, four or one signals in the 31P{1H} NMR were observed respectively. We attribute four signals in compound 3 to the presence of two diastereomers of the quinoxaline ligand and the one signals in compound 6. P-coupling through a P-Au-P bridge was observed in both 31P{1H} NMR spectra. In compound 3, Jpp was 0.12 ppm while in compound 6 it was 0.41 ppm. The presence or absence of the resonance structure of the P-C-C-P bridge influences Jpp coupling. Specifically, Jpp is smaller in the structure having the π electron bridge (complex 3), and Jpp is relatively larger in the structure where the π bridge is nonexistent (complex 6). The 1H-NMR and 13C{1H} NMR of compound 3 shows signals that can be attributed to diastereomer resonances. Furthermore, 2-dimensional COSY  and both phosphine and cyclometalated ligands  are shown in Figs UV-Vis spectra of representative complexes were measured in air-equilibrated pH 7.4 phosphate-buffered saline (PBS) or DMEM cell culture medium, which constitutes several reducing amino acids in high concentrations (mM range). The spectra of the complexes bearing phosphine  or metal-to-ligand charge-transfer (MLCT) character, which has been well-characterized by TDDFT calculations (vide infra). For example, in complex 7 there are two bands <300 nm, there is a high-energy band at 245 nm and another peak at 295 nm, which may be as a result of MLCT or LMCT transition73. There was no change in the absorbance profile of the gold(III) complex bearing cyclometalated and phosphine ligands (3) in PBS and minor changes in DMEM over the course of 48 h , shows a continuous decrease in absorption peaks at 248 and 337 nm over time in PBS. In contrast, complex 3, which is Au(III) showed no change in absorption bands. It can be deduced that the Au-P bonding in complex 2 is weakly stable in PBS compared to that of Au-P of complex 3. This is supported by computational results, in that, while the four Au-P bonds of complex 2 are ~2.5 Å, the two Au-P of complex 3 show shorter bond lengths of 2.38 and 2.5 Å, respectively, indicative of stronger bonds. The stability associated with 3, could be ascribed to both cyclometalated-Au bonding and adjacent Au-P bonds that enhance sigma-donation to the gold center. Complex 7, which is another Au(III) display significant changes to the absorption bands in DMEM. It appears that chloride, a good leaving group, is substituted by amino acids contained in DMEM74. On the contrary, it shows stability in PBS, this seems like PBS does not contain components to replace Cl of complex 7. Additionally, PBS contain 10 mM of NaCl that is likely to help stabilize the complex.Complex 75. The basis set used was SDD on Au atom, taking into consideration relativistic effects and the Pople-type 6–31 G was used for all other atoms. The geometry of the computed complexes was compared with the solved X-ray crystal structures to ensure the validity of the computations performed. The result is summarized in Fig. 1, two independent distances between Au atom and P atom were 2.22 and 2.23 Å, and the calculated values showed minimal discrepancy, with an error of 3%. Similarly, two independent bond distances between Au and Cl was 2.28~2.29 Å, whereas the calculated values were within ~2% error. Interestingly, the bond angle of P-Au-Cl, from the crystal structure of 1 was 171° and the corresponding calculated value was 171°. In the case of 2, the distorted structure of 2 had four P-Au bonds and its distances were all ~2.39 Å, and the calculated values were ~2.5 Å. Measured angles of three kinds of P-Au-P were 87.79°, 137.50°, and 106.48°, respectively, while the calculated values were 86.13°, 132.65°, and 111.58°. Unlike 1 and 2, compounds 4 and 5 possess dppe ligands, which also showed minimal discrepancy between the experimental and calculated bond distances or angles. For complex 4, the two gold metal atoms had two separate P-Au-P bonds, all four P-Au bond distances were 2.31 Å and the calculated values were ~2.38 Å. The X-ray structure revealed two P-Au-P angles as 166.8° and 177.8°, with the determined calculated angles were 169.1° and 171.0°, respectively. Complex 5 had a distorted structure like 2. The distance between the four P-Au distances were all 2.40 Å, while the calculated values was in the range of 2.35~2.36 Å, showing less than 3% error. The three P-Au-P angles were 86°, 117°, and 118°, and the calculated values were 91°, 128°, and 114°, respectively. Taken together, this shows that DFT calculations and the method employed can be considered suitable for providing accurate geometric information of gold complexes.To gain theoretical insight into the gold complexes under investigation, DFT calculations were conducted. We used Gaussian 09 and the B3LYP functional was employedTo corroborate peak assignments within the experimental absorption profiles and further provide insight into HOMO-LUMO transitions, we performed TD-DFT calculations.6 was compared to the calculated spectrum. The oscillator strength and molecular orbital (MO) contributions obtained from the theoretical calculation is summarized in Table 77. Furthermore, for complex 1, the contribution of HOMO-2 to LUMO was the largest, corresponding to a MLCT transition. The HOMO-2 is largely localized on the gold and the neighboring Cl atoms and the LUMO on the phenyl/pyrazine of quinoxaline. A similar charge transfer is seen in HOMO-LUMO transition for compound 1  and 5  and λ = 249 nm (HOMO-1 to LUMO + 1), which are charge transfer from the gold center to both quinoxaline ligands. For λ = 326 nm, the HOMO-LUMO transition represents a similar transfer from the gold center to the periphery of the molecule. For complex 5, electrons distributed around the metal migrate to neighboring phenyl groups: λ = 243 nm (HOMO to LUMO + 3) and λ = 292 nm (HOMO to LUMO). Similarly, for complex 5, the high energy is caused by MLCT of HOMO to LUMO + 3, and the neighboring λ = 292 nm (HOMO to LUMO) by gold center to the surrounding phenyl groups of the the dppe ligand. In contrast, complexes 3, 4, 6 and 7 show mainly LMCT transitions. For 3, high energy peaks (λ = 256 nm) were observed in quinoxaline-to-gold charge transfer (HOMO-3 to LUMO and HOMO to LUMO + 1) and low energy peaks were considered as transition from benzopyridine to gold center  complexes all display LMCT transitions and the Au(I) complexes display MLCT transition with the exception of 4. Overall, the study provide insight into the MO contributions of the experimentally obtained absorption profiles of these cytotoxic compounds.In Fig. d 1 Fig. . Additiod 5 Fig. . 2 has told Fig. . For 6, ion Fig. . Althougd6 and D2O. Given that stock solutions of the complexes were prepared in DMSO prior to biological, photophysical, or electrochemical evaluation DMSO-d6 was used. Consequently, all the biological evaluation is in aqueous base medium, thus studying their stability in D2O was appropriate. We measured the 1H-NMR of complexes 1–6 within a temperature range of 24–80 °C . The rigid chiral bisphosphine ligand may be the culprit for the observed distortion. Additionally, the bond angles C-P-Au were different for the methyl and tert-butyl group bonded to the phosphorus. For example, C9-P1-Au1 is 108.1(3), whereas C10-P1-Au1 is 112.6(3), indicative of a larger angle influenced by the bulkier tert-butyl fragment. Importantly, the dinuclear gold compound does not have a bond between the two gold atoms. Compound 2 crystallizes in the monoclinic, C2 space group which is a deviation from 1, despite the similar chiral ligand environment. The complex exhibits an unusually distorted square planar geometry for Au(I) species. The Au-P bond distances for both 1 and 2 are slightly invariant as we observe a longer bond distance for Au-P in 2, when compared to Au-P distances in 1. For example, complex 1, Au1–P1: 2.225(2) Å and Au2–P2: 2.236(2) Å, and for the complex 2, Au1-P1: 2.3889(14) Å, Au1-P4: 2.390(2) Å, Au1-P2: 2.3928(19) Å, and Au1-P3: 2.3964(15) Å, respectively. The distorted square planar geometry of 2, contribute to the longer Au-P bond distances than that of 1. We note that the Au-P bonds for the structure of 4 is comparable to that of compound 2 but the Au-P bond distances for 5 are longer, averaging 2.406(8) Å. This is consistent with the fact that distorted gold(I) complexes such as 2 and 5 exhibit slightly longer Au-P distances than their dinuclear counterparts, 1 and 4. The cone angle of DPPE to the Au center in 5 are smaller (P1-Au1-P2 85.97(3) Å) in comparison to the -(-)-2,3-bis(t-butylmethylphosphino) quinoxaline ligand in 2 (P1-Au1-P2 87.91(5) Å).Single crystals of four complexes out of the six compounds studied were obtained and the crystal structures were determined by X-ray crystallography. Crystal structures with optimized structures for 4) as supporting electrolyte at a Pt electrode, using CH-600D potentiostat equipment. One reversible reduction event was observed for the gold(I) complex, 1 at a potential of −1.20 V  complex that may lead to two closely distinct redox events. The result and the absence of metallic gold at the platinum working electrode led to the conclusion that the overall reduction for this class of Au(I) complexes is ligand-centered rather than metal-centered78.To elucidate the reduction and oxidation properties of these gold complexes, we measured the cyclic voltammetry in DMSO with sodium perchlorate  complexes bearing DPPE ligands were investigated. Complex 4 shows irreversible reduction peak at −0.84 and −1.54 V  complexes, 3 and 6 in DMSO using cyclic voltammetry. As shown in Fig. 3. In addition, a corresponding oxidation event was observed at −1.27. 6 showed two reversible reductions at −1.01 and −1.37 V, and the paired oxidation events at –0.86 and −1.31 V were observed, respectively /Au(0) reduction in aqueous solutions due to disproportionation of Au(I) in aqueous solution82. Our observation lean more towards the former with an initial Au(III)/Au(I) reduction for 3 and 6.The electrochemical behavior of 4 V Fig. . Similarnts Fig. . The eveely Fig. . Typical1–6) described in this report in a reaction with bovine serum albumin (BSA) was performed under physiological conditions. Taking advantage of the intense absorption bands of the gold complexes and BSA, we monitored the progress of the reaction using 1:1 ratio of BSA and buffered solutions of each gold complex over 24 h. Serum albumin is a major soluble protein component present in the circulatory system and has many physiological functions83. Importantly, BSA acts as a carrier for various pharmacological agents. It must be noted that BSA has been extensively studied, and shares homology with human serum albumin (HSA)84. Often, gold compounds bind methionine and cysteine residues in BSA via the sulfur atoms. The inherent absorption peaks for complexes 2–6 were minimally affected by the addition of the BSA solution over a 24 h period  compounds with cyclometalated ligands but different bisphosphine ligands. Interestingly, none of the peaks associated with the complexes or BSA changed, indicative of stability in the presence of BSA over 24 h. While the shoulder peak at 300 nm disappeared in the case of complex 4 (LMCT), the BSA peak at 250 nm was unmodified. Complex 5 on the other hand did not display any alteration in its peak. In general, there was no indication of the formation of metallic gold as no brown precipitate formed in any of the reaction over the duration of the experiments. Using HPLC  complexes with serum albumin under similar experimental conditions showed varied stability of the gold complexes in the presence of BSA. This result has important implications for the pharmacological activity of these gold complexes, in that they can avoid premature deactivation until they reach their target and also reduce off-target effects.Spectrophotometric investigations of the gold complexes  summarized in Table 1–6 displayed high nanomolar to low micromolar cell killing which are 2–10 folds better than cisplatin. None of the gold-phosphine complexes display cross-resistance evidenced by indifferent toxicities in cisplatin resistant cells, including the well-characterized high grade-serous ovarian cancer (HGSOC) cell line, OVCAR8, which demonstrate the example of high potency of these novel Au complexes in clinically relevant tumor cells. Generally, the gold compounds studied are slightly less potent toward RPE-Neo cells, indicative of selective toxicity for cancerous cells compared to healthy cells. Compounds 2 display ~8–40-fold higher potency in cancer cells than the normal RPE-Neo cell line.The antiproliferative properties of these gold complexes were evaluated in a panel of cancerous cell lines using crystal violet assay for H460 and OVCAR8. We used an ATP-dependent luminescence cell assay, cell titre glo, for K562 cells. To extend the therapeutic utility of these novel drug candidates, we performed cytotoxicity studies with normal retinal pigment epithelium, RPE-NEO. Auranofin and cisplatin were used as controls. We obtained dose-response curves from the cell viability experiments and subsequently derived ICTable 1–6 and auranofin as well as subcellular distribution of Au for 1 and 3. OVCAR8 cells were incubated with the gold compounds (5 μM) for 15 h. The Au content was measured by using ICP-OES, calibrated with HAuCl4 standard. Differential whole cell uptake was observed for all compounds with neutral complexes, auranofin and 1 displaying the highest uptake respectively and 6 taken up the least  over mononuclear gold complexes bearing chiral or achiral phosphine ligands respectively. It is likely that the dinuclear gold increases lipophilicity of the agents. Additionally, the use of chiral QuinoxP ligands have a higher lipophilic character over the dppe ligands. We evaluated 1 and 3, which bear the same chiral ligand but different oxidation states in a series of biological experiments and although IC50 values do not show significant differences, apoptosis assay (vide infra) reveal a much higher early to late-stage apoptosis for 1, which has a higher cellular uptake than 3. Following the incubation of OVCAR8 cells with 1 or 3, we isolated the nuclear component from the cytoplasm and the residual cell pellet. As shown in Fig. 1 or 3 was localized predominantly to the cytoplasmic fraction at 200 pmol/106 cells concentration. In contrast, a lower localization within the nuclear fraction at 15 pmol/106 cells was observed for both 1 and 3. The similar Au concentration in the subcellular fractions despite the different structural scaffolds may be the clue to the similar growth inhibition observed. The concentration found within the cell residue (pellet) was similar to that of the nuclear fraction, within margin of error. These results demonstrate that the new Au constructs largely localize and may target cytoplasmic proteins owing to their lipophilic chiral ligands with minimal potential to target genomic DNA.To gain insight into the intracellular behavior of the gold reagents, we studied the whole cell uptake of 1 or 3, the compounds were examined for apoptotic effects. Mitochondria dysfunction and ER stress can result in apoptosis. Characteristically, cells undergoing apoptosis have associated cell membrane disorientation. This leads to efficient phosphatidylserine residue translocation from the interior of the cell to the exterior membrane, which can be readily detected by Annexin V molecules. We performed a dual-stain experiment involving FITC-labeled Annexin V and propidium iodide (PI) for flow cytometry analysis. The HGSOC cell line, OVCAR8, were treated with 1, 3, auranofin or cisplatin for 72 h. We observed a large population occurring as late-stage apoptotic cells for the Au(I) compounds, i.e. 1 and auranofin. In general, all the Au complexes induce significant apoptosis over cisplatin as shown in Fig. in vitro potency when compared with the conventional platinum(II)-based agent, cisplatin.To further understand the cellular responses evoked by 3, as measured by the fluorescence of dihydrorhodamine 123 using flow cytometry. Together, the study suggests the ability of these gold complexes to modulate intracellular ROS.Suspicious of mitochondria dysfunction and ROS production as primary inducers of cell death, we conducted intracellular reactive oxygen species (ROS) experiments using human cancer cell line, OVCAR8 Fig. . Mitocho1–6 on intracellular ROS, mitochondrial membrane potential studies were conducted to monitor mitochondrial insults. Notably, 1 and 2, largely depolarized the mitochondria membrane by measuring the fluorescence of rhodamine 123 using flow cytometry -phosphine complexes as effective anticancer agents.The synthesis and characterization, electrochemical, and biological evaluation of six gold complexes bearing chiral and achiral bisphosphine ligands were investigated. All compounds display potent cytotoxic effects in cancer cells over normal cells. Particularly, the gold compounds described show high antiproliferative activity in a panel of cell lines including the clinically relevant HGSOC OVCAR8 cell line, ranging from 2–10-fold lower IC1H, 13C, 31P, COSY, and HSQC NMR spectra were recorded on a Varian Unity 400/500 NMR spectrometer with a Spectro Spin superconducting magnet in the University of Kentucky NMR facility. Chemical shifts in 1H, 13C, COSY, and HSQC NMR spectra were internally referenced to solvent signals , and those in 31P NMR spectra were externally referenced to 85% H3PO4 in D2O (δ = 0 ppm). Electrospray ionization mass spectrometry (ESI-MS) was performed on an Agilent Technologies 1100 series liquid chromatography/MS instrument. High-resolution mass spectra (HRMS) were obtained by direct flow injection (injection volume = 5 or 2 μL) ElectroSpray Ionization (ESI) on a Waters Qtof API US instrument in the positive mode . Typical conditions are as follows: capillary = 3000 kV, cone = 35 or 15, source temperature = 120 °C, and desolvation temperature = 350 °C. In addition to spectroscopic characterization, bulk purity of all new compounds was assessed by combustion elemental analysis for C, H, N. Elemental analysis was carried out at the microanalysis lab at University of Illinois Urbana Champaign using Perkin Elmer 2440, Series II with a combustion temperature of ~2000 °C and accuracy of 0.3% abs. Reactions were monitored using aluminum backed silica-gel thin-layer chromatography (TLC) plates  and visualized under low-wavelength light (254 nm) or stained with iodine on silica for visualization with the naked eye. Purification of reactions was performed using silica-gel  chromatography. The CombiFlash® Rf + Lumen, Teledyne ISCO was used purification of some compounds. Quantification of elemental gold in samples was performed using ICP-OES at the University of Kentucky Energy Research and Training Laboratory. Flow cytometry experiments were performed at the University of Kentucky flow cytometry core facility. Quantum chemical calculations using Gaussian was performed on the University of Kentucky high performance computing (HPC) facility.Auranofin and Cisplatin were purchased from Santa Cruz Biotechnology Inc. and Strem, respectively and used without further purification. All other reagents were purchased from Strem, Sigma Aldrich, or Alfa Aesar and used without further purification. All reactions were carried out under normal atmospheric conditions. Deuterated solvents were purchased from Cambridge Isotope Laboratories . digold(I) (1) Under normal atmospheric conditions, in a 10 mL scintillation vial was placed -(-)-2,3-bis(t-butylmethylphosphino)quinoxaline . Chloroform (2 mL) was added and the solution (golden) was stirred at room temperature. To the solution was added solid white Au(tht)Cl . The solution turned brick red instantly. The solution was stirred for about 15 min and monitored by TLC in 100% chloroform. The solution was passed through celite and solvent allowed to evaporate under vacuum. The residue was washed with diethyl ether and filtered. The solid was dried in vacuo to obtain a light purple solid. Yield: 21 mg, 57.1%. 1H NMR  δ 8.21 , 8.01 , 2.19 , 1.43 . 13C NMR  δ 153.36, 153.16, 152.96, 152.74, 152.53, 140.13, 140.09, 140.05, 133.55, 129.39, 35.93, 35.89, 35.74, 35.73, 35.57, 35.53, 28.06, 28.02, 28.00, 11.98, 11.96, 11.79, 11.75, 11.58, 11.56. 31P NMR  δ 29.02. HRMS (ESI) (m/z): calcd. for C18H28Au2Cl2N2P2 [M + Na] 821.0334, found: 821.0362. Anal. Calcd. for C18H28Au2Cl2N2P2: C, 27.05; H, 3.53; N, 3.51. Found: C, 27.13; H, 3.77; N, 3.89.Bis-gold(I) chloride (2) Under normal atmospheric conditions, in a 25 mL round bottom flask was placed -(-)-2,3-bis(t-butylmethylphosphino)quinoxaline . Chloroform (6.0 mL) was added and the solution (golden) was stirred at room temperature. To the solution was added solid white Au(tht)Cl . The solution turned brick red instantly. The solution was stirred for about 15 min and monitored by TLC in 5% MeOH in DCM. A purple precipitate was obtained. Yield: 26 mg, 23.0%. 1H NMR  δ 8.25 , 7.99 , 2.01 , 1.28 . 31P NMR  δ 13.05. HRMS (ESI) (m/z): calcd. for C36H56AuN4P4 [M-Cl]+ 865.3121, found: 865.3104. Anal. Calcd. for C36H56AuClN4P4.0.3CH2Cl2: C, 47.05; H, 6.16; N, 6.05. Found: C, 47.18; H, 6.27; N, 5.90.(2-benzoylpyridine)[quinoxaline]gold(III) dichloride (3) Under normal atmospheric conditions, cyclometalated Au(III)Cl2  was placed in a 25 mL round bottom flask and 5 mL of chloroform was added, the solution turned white. -(-)-2,3-bis(t-butylmethylphosphino)quinoxaline  was added, the solution turned golden peach color. The solution was stirred for about 2 hours. The solution was monitored by TLC in 5% MeOH in DCM and Au starting materials disappeared. The product was separated by flash silica-gel chromatography with 5% MeOH in DCM as eluent. A bright purple precipitate was obtained. Yield: 35.8 mg, 73.6%. 1H NMR  δ 8.80–8.75 , 8.48 , 8.40–8.20 , 8.18–8.01 , 8.01–7.92 , 7.92–7.83 , 7.76–7.62 , 7.41 , 2.68 , 2.47 , 2.38 , 2.02 , 1.48–1.36 , 1.15–1.05 . 13C NMR  δ 198.15, 198.11, 157.15, 155.89, 155.29, 150.28, 149.66, 149.49, 149.00, 148.81, 148.72, 141.97, 141.90, 139.66, 139.6, 137.92, 137.89, 137.37, 136.95, 136.88, 135.37, 135.27, 135.14, 135.02, 134.54, 134.45, 134.18, 129.98, 126.79, 126.52, 126.34, 126.04, 124.58, 124.47, 54.92, 37.91, 37.68, 36.97, 36.73, 27.11, 27.09, 26.53, 26.51, 26.48, 26.45, 25.34, 25.32, 6.43, 6.10, 5.62, 5.01, 4.66, 4.37. 31P NMR  δ 50.35 , 49.39 , 43.82 , 37.83 . HRMS (ESI) (m/z): calcd. for C30H36AuClN3OP2 [M-Cl]+ 748.1688, found: 748.1685. Anal. Calcd. for C30H36AuCl2N3OP2.0.5CH2Cl2.0.8(CH3CH2)2O: C, 45.67; H, 5.12; N, 4.74. Found: C, 46.03; H, 4.96; N, 4.40.Bis]]digold(I) diperchlorate (4) Under normal atmospheric conditions, HAuCl4·3H2O , 1,2-Bis(diphenylphosphino)ethane, DPPE, , and NaClO4  were placed in a 100 mL round bottom flask and 40 mL of MeCN was added. The reaction solution was stirred and refluxed overnight. And then it was filtered and the solvent was allowed to evaporate under vacuum. A white precipitate was obtained. Yield: 146.8 mg, 20.8%. 1H NMR  δ 7.80–7.09 , 2.55 . 13C NMR  δ 133.22, 132.03, 130.34, 129.33, 128.85. 31P NMR  δ 21.23. HRMS (ESI) (m/z): calcd. for C52H48Au2ClO4P4 [M-ClO4]+ 1289.1523, found: 1289.1545.Bis]gold(I) perchlorate (5) Under normal atmospheric conditions, HAuCl4·3H2O , 1,2-Bis(diphenylphosphino)ethane, DPPE, , and NaClO4  were placed in a 100 mL round bottom flask and 40 mL of MeCN was added. The reaction solution was stirred at room temperature overnight. And then it was filtered and the solvent was allowed to evaporate under vacuum. A white precipitate was obtained. Yield: 230 mg, 41.4%. 1 H NMR  δ 7.88–7.11 , 2.55 . 13C NMR  δ 133.25, 132.03, 130.34, 128.85, 27.55. 31P NMR  δ 21.24. HRMS (ESI) (m/z): calcd. for C52H48AuP4 [M-ClO4]+ 933.2372, found: 933.2385(2-benzoylpyridine)]gold(III) dichloride (6) Under normal atmospheric conditions, cyclometalated Au(III)Cl2  was placed in a 25 mL round bottom flask and 5 mL of chloroform was added. 1,2-Bis(diphenylphosphino)ethane, DPPE,  was added and the reaction solution was refluxed for 4 hours. The reaction was monitored by TLC in 5% MeOH in DCM, the target compound showed an Rf ~0.3. After TLC. The product was separated by flash silica-gel chromatography with 5% MeOH in DCM as eluent. Yield: 19.4 mg, 38.9%. 1H NMR  δ 8.59 , 8.22–8.06 , 7.95 , 7.83–7.73 , 7.72–7.34 , 3.79 , 3.18 . 13C NMR  δ 193.98, 151.88, 148.73, 141.73 , 137.60 , 134.32, 133.47–132.48 (m), 131.54, 129.69, 129.11, 127.99, 124.55, 120.11, 119.24, 117.95, 117.10, 79.20, 24.49, 22.09. 31P NMR  δ 27.87, 27.46. HRMS (ESI) (m/z): calcd. for C38H32AuClNOP2 [M-Cl]+ 812.1313, found: 812.1315. Anal. Calcd. for C38H32AuCl2NOP2: C, 53.79; H, 3.80; N, 1.65. Found: C, 53.51; H, 3.90; N, 1.46.Dichloro(2-benzoylpyridine)gold(III) (7) This procedure was adapted from previous reports in the literature95. 1H NMR  δ 9.48 , 8.56 , 8.37 , 8.14–8.05 , 7.76 , 7.73–7.65 , 7.48 . LRMS : calcd. for C12H8AuCl2NO [M+] 448.96, found: 449.00.1–7 were prepared in DMSO. For studies with PBS and DMEM, the gold solutions were diluted with the respective diluent (PBS or DMEM) to achieve a final gold concentration of 25 μM or 50 μM. Background scans were performed with the corresponding DMSO or solvent mixture. For the reactivity experiment with BSA, the sample solution was prepared by the above-mentioned method, and BSA was separately dissolved in PBS to prepare 1 mM stock solution and diluted with PBS. The sample solution was mixed with the BSA solution immediately before the measurement. Single scan or time-based scanning was performed as needed. The UV-Vis spectrometer used was a Shimadzu UV-1280 model.Stock solutions (1 mM) of complexes 4, which were purchased  and used without further purification. All complexes (1−6) were dissolved in DMSO in the concentration of 1.0 mM, followed by addition of 0.01 M NaClO4 as a supporting electrolyte. Electrochemical measurements were performed at ambient temperature using CH-600D potentiostat  for cyclic voltammetry. The voltammetric measurements were performed in a three-electrode cell containing a platinum working electrode, a non-aqueous Ag/AgCl reference electrode and a platinum wire as counter electrode.Cyclic voltammetry was performed in dimethyl sulfoxide (DMSO) with NaClO3 cells/well) and were incubated with RPMI 1640 supplemented with 10% FBS (150 μL) for 24 h and at 37 °C. They were then treated with cisplatin, auranofin, or 1–6 at increasing concentrations for 72 h from stock solutions following serial dilutions. Cisplatin stock solution was prepared using PBS and the gold compounds were prepared in DMSO. Thereafter, cellular viability was assessed via the established crystal violet colorimetric assay. In brief, crystal violet reagent  was added to each well and allowed to incubate with cells for 30 min. The plates were washed with water by running them under a gentle flow of tap water. The plates were air dried and dissolved with methanol (100 μL/well) and the plates were rocked for an additional 10 min. Measurements of absorbance were subsequently performed using a Genios plate reader at 570 nm (peak absorbance). All experiments were conducted in triplicate. For K562 cells, a cell density of 4,000 cells/well was used and cellular viability was assessed after treatment, using the established luminescent cell titre glo assay.Various established human ovarian, leukemia and lung cancer cell lines were seeded in 96-well plates (2 × 106) were seeded in a 6-well plate and incubated for 15 h at 37 oC. Cells were then incubated with the test compounds (5 μM) in fresh RPMI 1640 medium (10 mL) and subsequently incubated for a given period of time (~24 h) at 37 °C. The medium was then removed and cells were collected via trypsinization. Cells were then washed with PBS (1 mL x 3). The cells were digested by adding 0.5 mL of concentrated HCl and briefly placing them on an agitator. Cells were then transferred to a new tube containing 4.5 mL of DI water. The gold content was analyzed by ICP-OES to obtain the whole cell uptake after quantification.OVCAR8 cells , harvested, and centrifuged. The cell pellet was suspended in an appropriate volume of PBS to obtain a homogeneous suspension (100 μL). The nuclear and cytoplasmic extraction kit  was used to extract the separate cytoplasmic, nuclear, and pelleted fractions. The fractions were mineralized with 70% HNO3 and then heated at 95 °C for 10 min. The gold content was analyzed by ICP-OES. Cellular gold levels were expressed as pmol of Au per million cells. Results are presented as the mean of three determinations for each data points.To measure the intracellular cellular uptake, ~1 million OVCAR8 cells were treated with 5 μM compounds 5 cells/well) and were treated with PBS, 1, or 3 for 24 or 48 h at a concentration of 2 µM. The cells were re-suspended in ice-cold PBS, fixed with 70% ethanol in PBS at 4 °C overnight, subsequently washed with ice-cold PBS (x2), and then incubated with RNase A (1 μg/mL) for 20 min at 37 °C. They were then stained with PI (10 μg/mL for 30 min in the dark) and their DNA content and cell cycle distribution were measured, using a FACSCalibur flow cytometry  and as determined with ModFit software.OVCAR8 cells were seeded in 6-well plates (2 × 105 cells/well), treated with PBS, cisplatin, auranofin, 1 or 3 at a fixed Au concentration (2 µM) for 48 h, and harvested by trypsinization. The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was used to determine the fraction of cells  that underwent apoptosis, using fluorescence-activated cell-sorting sorting  and by following the manufacturer’s protocol. Data were analyzed using FlowJo software.OVCAR8 cells were seeded in 6-well plates  was dissolved and diluted with PBS to a final concentration of 0.005 mg/mL. The solution was added (0.5 mL) to the cells re-suspended, and incubated for 30 min. Cells were pelleted by centrifugation, washed with 1 mL of PBS, and re-suspended in 0.5 mL of PBS for flow cytometry analysis.OVCAR8 cells  were plated in 6-well plates taking into account respective controls  and test compounds. After overnight incubation of cells at, 37 °C and 5% COOVCAR8 cells  were plated on a 6-well plates. The cells were then treated with 5 μM gold compounds and incubated for 24 h and 48 h at 37 °C, after which media was removed and cells were washed with PBS. The cells were scraped into SDS-PAGE loading buffer (64 mM Tris-HCl (pH 6.8)/9.6% glycerol/2% SDS/5% β-mercaptoethanol/0.01% bromophenol blue) and incubated at 95 °C on a heat block for 10 min. The samples were cooled and stored at −20 °C until ready for use. Whole cell lysates were resolved by 4–20% sodium dodecylsulfate polyacrylamide gel electrophoresis  followed by electro transfer to a polyvinylidene difluoride membrane, PVDF (350 mA for 1 h). Membranes were blocked using 5% (w/v) bovine serum albumin (BSA) in PBST (PBS/0.1% Tween 20) and incubated with specific primary antibodies  overnight at 4 °C. On the following day, after washing with PBST (3 × 5 mL), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies  in fresh BSA blocking solution. Immune complexes were detected with the ECL detection reagent (BioRad) and analyzed using a BioRad imager fitted with a chemiluminescence filter. Intracellular ROS using murine polymorphonuclear neutrophils.1, 2, 4, and 5 were grown at room temperature by vapor diffusion of diethyl ether into a DMF or DCM solutions of each complex. Suitable crystals were selected by microscopic examination through crossed polarizers, mounted on a fine glass fibre in polyisobutane oil, and cooled to 90 K under a stream of nitrogen. A Bruker D8 Venture diffractometer with graded-multilayer focused MoKα X-rays (λ = 0.71073 Å) was used to collect the diffraction data from the crystal. The raw data were integrated, scaled, merged and corrected for Lorentz-polarization effects using the APEX3 package98. Space group determination and structure solution and refinement were carried out with SHELXT and SHELXL100, respectively. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were placed at calculated positions and refined using a riding model with their isotropic displacement parameters (Uiso) set to either 1.2Uiso or 1.5Uiso of the atom to which they were attached. The structures, deposited in the Cambridge Structural Database, were checked for missed higher symmetry, twinning, and overall quality with PLATON101, an R-tensor102, and finally validated using CheckCIF101. See Tables −) for 4 and 5, which make them crystallographically distinct from similar published structures.Crystals of 2.All ovarian cancer cells (OVCAR8), lung cancer cells (H460), and the leukemia cell line (K562) were maintained in the Roswell Park Memorial Institute (RPMI) 1640 medium. With the exception of H460 which was supplemented with 15% heat inactivated fetal bovine serum (FBS) and 10% glutamine, all other cell lines were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were grown at 310 K in a humidified atmosphere containing 5% CO75. Density functional theory (DFT) and time dependent density functional theory TD-DFT with Becke’s three-parameter hybrid functional with the correlation functional of Lee, Yang, and Parr (B3LYP)103 was used to calculate the equilibrium geometries and vibrational frequencies of 1–6. Stuttgart-Dresden effective-core-potential basis set (SDD) for Au and 6–31 G for C, H, N, O, P, and Cl were employed for these calculations. A vibrational analysis was accomplished to identify the nature of each optimized stationary points no imaginary frequencies were found. Optimized XYZ coordinates for all calculated molecules are attached on Table All calculations were performed using the Gaussian 09 package1–7, and data regarding all X-ray crystallography, molecular biology. Crystal structures of 1 (CCDC 1817925), 2 (CCDC 1817924), 4 (CCDC 1852670), and 5 (CCDC 1852671) have been deposited at the Cambridge Crystallographic Data Centre.Supporting Information is available free of charge on the Nature Publications website. Experimental details, characterization of compounds ESI


Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium  Their discovery and characterization (Klebsiella pneumoniae and belongs to class B.Dye-decolorizing peroxidases (DyPs)rization , 2 led trization . These mrization . Phylogerization . Except Rodococcus jostii (Pseudomonas putida (Escherichia coli (Vibrio cholerae (Enterobacter lignolyticus (e.g. Reactive Blue 19), Mn(II) ions, or even lignin model compounds  exhibitompounds , 10–13. helatase , or lignhelatase . However•+) (Reaction 2) or a distinct amino acid radical (aa•+) (Reaction 3). Additionally, water is released.In all heme peroxidase (super)families , the per2O2 is still under dispute. In B-class DyPs, both the Arg -type Compound II  , Compountii RHA1  and P. p. putida ) and thes, ElDyP ) have bes, ElDyP , 17. Likpound II .aK = 13.8) porphyrin π-cation radical of very poor oxidation capacity, whereas substitution of Asp-143 significantly increases the two- and one- electron oxidation capacity of Compound I. Furthermore, we demonstrate the formation of an oxoiron(IV)-type Compound II for both WT KpDyP and the variant D143A.Here, we hypothesize that the distal aspartate acts as proton acceptor during heterolytic cleavage of hydrogen peroxide. This hypothesis is based on the facts that (i) B-class DyPs typically exhibit pH optima of enzymatic activity below pH 7 and deprotonation of a guanidinium group (p = 13.8)  per se bironment  and (ii)2O2, we aimed at characterization of the solution and crystal structures of WT KpDyP and its variants D143A, R232A, and D143A/R232A. Consequently, these proteins were expressed heterologously in E. coli and purified by affinity and size-exclusion chromatography (SEC) as described under “Experimental procedures.” Typically, 60–80 mg of WT and variant heme protein per liter of culture broth were obtained. HPLC-SEC combined with multiangle light scattering confirmed the presence of pure homodimeric WT and variant proteins (2 × 35 kDa) (data not shown), which is in accordance with previously published B-class DyPs. The average Reinheitszahl (ASoret/A280) was found to be 2.1 (WT KpDyP), 1.7 (D143A), and 2.7 (R232A and D143A/R232A), respectively. Upon using the pyridine-hemochromogen assay (m−1 cm−1 (WT KpDyP and D143A) and 240,000 m−1 cm−1 (R232A and D143A/R232A), respectively.As a basis for elucidation of the role of Asp-143 and Arg-232 in heterolytic cleavage of Hen assay , the extA and B) depict the UV-visible and EPR spectral signatures of WT KpDyP and the variants. At pH 7.0, all proteins are in the high-spin (HS) state. WT KpDyP exhibits UV-visible bands at 405 nm (Soret band) together with a small shoulder at 385, 507, and 539 nm (Q-bands) and 640 nm (charge transfer (CT) band). Except for the broad Soret maximum at 400 nm, the spectrum of the D143A variant is identical with that of the WT protein. In the variants R232A and D143A/R232A, the CT band is blue-shifted to 630 nm, whereas the other bands are almost WT-like . Except for the D143A variant, no spectral changes were observed between pH 4.5 and pH 10. In the case of D143A, an alkaline transition occurred (paK = 8.6), resulting in a species with Soret maximum at 410 nm and additional bands at 540, 576, and 610 nm, indicative of the presence of a HS hydroxo ligand  or a mixed population of HS and LS hydroxo ligated heme iron.KpDyP was further investigated using low-temperature continuous-wave (CW) EPR spectroscopy. The experimental spectra of ferric WT KpDyP and its distal side variants at neutral pH (B) clearly display the presence of HS . On the other hand, the EPR spectra can also be simulated assuming an apparent S = ½ system with effective geff tensor. Both approaches were assumed here, to allow comparison with literature data . The features and g-anisotropy  of this EPR signal .The electronic CD (ECD) spectra in the far-UV  and near-UV region (250–350 nm) of the four proteins were almost identical, suggesting that the mutations did not change the overall secondary and tertiary structure. By contrast, ECD spectra in the visible region showed differences, again reflecting altered heme cavity architectures in the four proteins. At pH 7.0, WT ncreased C.E°′) of the Fe(III)/Fe(II) redox couple of KpDyP and its catalytic variants by spectroelectrochemical titration at pH 7.0. Upon reduction of ferric to ferrous WT KpDyP, the Soret peak shifts from 405 to 435 nm. black and red lines, respectively) as well as the equilibrium spectra of WT KpDyP (gray lines) at seven different applied potentials in the optical transparent thin-layer spectroelectrochemical (OTTLE) cell . The calculated reduction potential E°′ for the Fe(III)/Fe(II) couple, determined from the corresponding Nernst plot (inset), was calculated to be −0.350 ± 0.010 V. Removal of Asp-143 slightly increases the standard reduction potential (D143A: −0.330 ± 0.010 V) compared with the WT protein, whereas E°′ is almost unaffected by elimination of R232 (R232A: −0.347 ± 0.010 V). The E°′ value of D143A/R232A was calculated to be −0.299 ± 0.010 V.Next, we probed the standard reduction potential  because DyPs are often described for optimum peroxidase activity at acidic pH (pH <3.0). As is appropriate for a cytoplasmatic protein, WT KpDyP was found to be most stable between pH 5.5 and pH 7.0 (D). Temperature-mediated unfolding shows one endotherm that can be best fitted by assumption of a non-two-state transition with two consecutive irreversible unfolding events at mT values of ∼58 and ∼60 °C at pH 5.5, which cannot be separated. All variants exhibited decreased thermal stability but followed a similar unfolding profile and pH dependence. Typically, the mT values of the mutants were lowered by 5–10 °C following the hierarchy WT > D143A > R232A > D143A/R232A. Below pH 4, WT KpDyP was too unstable and aggregated upon heating.Finally, we probed the thermal stability of WT d pH 7.0 D. TemperKpDyP, we probed the kinetics and thermodynamics of the formation of the cyanide complex of KpDyP and the three variants, because cyanide binding mechanistically mirrors Compound I formation. Cyanide, which at most biological pH levels is protonated (paK = 9.14), binds as an anion to Fe(III). Thus, HCN and H2O2 need to be deprotonated by a distal base. Thereby, deprotonation of HCN mimics the committing step of H2O2-mediated Compound I formation. Upon the addition of cyanide, high-spin WT and KpDyP variants are converted to a low-spin complex with a Soret maximum at 420 nm. Spectrophotometric titration of KpDyP revealed that the dissociation constant (DK) of the WT protein is very high (900 ± 40 μm). Elimination of Arg-232 increased DK (2700 ± 370 μm). In the double variant, DK was determined to be 136 ± 13 μm) (data not shown). By contrast, binding affinity of cyanide was significantly increased upon elimination of the distal aspartate (D143A: 20 ± 12 μm derived from pre-steady-state kinetics).Before investigating Compound I formation of kon, of the reaction between WT KpDyP and cyanide was determined to be (7.6 ± 0.2) × 104m−1 s−1 . By contrast, cyanide binding to D143A strongly depended on pH, ranging from (5.9 ± 0.6) × 102m−1 s−1 at pH 5.0 to (2.3 ± 0.2) × 105m−1 s−1 at pH 8. At pH 7, kon of D143A was almost WT-like ((4.6 ± 0.6) × 104m−1 s−1). Upon elimination of Arg-232, cyanide binding was drastically reduced ((13 ± 1) m−1 s−1, pH 7.0), whereas kon for the double variant was calculated to be (6.1 ± 0.4) × 103m−1 s−1 at pH 7.0 . Compound I is characterized by a Soret maximum at 397 nm (∼50% hypochromicity compared with ferric KpDyP) and formation of an additional band at 648 nm. The reaction is monophasic (kapp = (6.2 ± 0.2) ± 106m−1 s−1 at pH 7.0) with clear isosbestic points at 430 and 550 nm (A). Compound I is already formed with equimolar H2O2, and its UV-visible spectrum remains unchanged for minutes, whereas at high excess of H2O2 (>1 mm), heme is degraded within seconds, as reflected by loss of absorbance and generation of a peak at 670 nm, which might indicate biliverdin formation (KpDyP is pH-independent between pH 4 and pH 9 (not shown).Next, we focused on the role of Asp-143 and Arg-232 in Compound I formation. Oxidation of WT low mode A. Compoud 550 nm A. Compouormation . SimilarB–D). However, a ∼400-fold stoichiometric excess of H2O2 is necessary to obtain full hypochromicity at the Soret band, and the apparent bimolecular rate constants are diminished by 3 orders of magnitude (insets to C and D). Moreover, the variants R232A (kapp = (2.2 ± 0.1 × 103m−1 s−1 at pH 7.0) and D143A/R232A (kapp = (4.5 ± 0.2 × 103m−1 s−1 at pH 7.0) are significantly more prone to heme degradation compared with the WT protein. Heme degradation is reflected by the biphasic kinetics of Compound I formation, with the second slow phase representing bleaching of the prosthetic group. In the case of D143A, Compound I formation (kapp = 1.9 ± 0.1 × 103m−1 s−1 at pH 7.0)  is followed by a slow H2O2-dependent conversion (kapp = 40 m−1 s−1) to a species reminiscent of classical oxoiron(IV)-type Compound II with absorbance maxima at 417, 527, and 555 nm . Similar to cyanide binding, the kapp values of Compound I formation of the variant D143A increase between pH 5.0 (1.4 ± 0.1 × 102m−1 s−1) and pH 8.0 (8.6 ± 0.1 × 103m−1 s−1). By contrast, Compound I formation of R232A slightly increases with decreasing pH.Upon the addition of hydrogen peroxide to the variants, similar spectral transitions were observed , B–D. Ho pH 7.0) B, inset 2O2 to WT KpDyP (bottom). The corresponding CW EPR spectrum exhibits radical-type signals and some residual HS Fe(III). Closer inspection of the former (inset to bottom panel) reveals that this signal is composite, displaying two features with a different saturation behavior . The most intense peak at g = 2.00 is in good agreement with the porphyrin π-cation radical observed in horseradish peroxidase  . A similar behavior is observed after H2O2 activation of DyP from Thermomonospora curvata  , 28. Thi curvata . Multifre.g. hydroxylation, dehalogenation, N-oxidation, etc.) did not yield any results.Next, we probed the enzymatic activity of KpDyP and investigated the spectral features of the involved redox intermediates of this heme peroxidase. WT KpDyP exhibits very poor overall peroxidase activities. Oxidation of bulky dyes like RB19, reactive black 5, indigo carmine, and acid red 183 was extremely slow. This also applies to conventional peroxidase substrates like guaiacol, pyrogallol, 3,3′,5,5′-tetrametylbenzidine (TMB), catechol, hydroquinones, tyrosine, and ascorbate (<0.5 units/mg) (not shown). Only ABTS was an exception, but also its oxidation is modest (∼40 units/mg). A search for alternative enzymatic activities (KpDyP and the D143A variant with various putative one- and two-electron donors in the multimixing stopped-flow mode (KpDyP Compound I (formed with a 2-fold stoichiometric excess of H2O2), whereas iodide reacted only to a limited extent. Thiocyanate mediates the slow conversion of WT Compound I directly to the ferric state with isosbestic points at 356, 452, and 553 nm (A). The time traces monitored at 386 nm , 405 nm (Soret maximum of ferric state), and 420 nm (isosbestic point between Compound II and ferric state) are biphasic and show an initial lag phase followed by slow formation of the ferric state mediated by 1 mm thiocyanate. The lag phase may reflect cycling due to excess of H2O2, as its length increases with increasing excess of H2O2. No lag phase is observed when substoichiometric concentrations of H2O2 are used.Finally, we have probed the direct reaction between preformed Compound I of WT low mode . Regardid 553 nm A. The tiB). Compound I was formed with a 500-fold stoichiometric excess of H2O2. Upon reaction with thiocyanate, ferric D143A was formed rapidly and directly with clear isosbestic points at 432, 455, and 538 nm. Fig. 6B shows a representative time trace of the reaction between D143A Compound I and 10 μm thiocyanate. Due to the excess of H2O2 necessary for Compound I formation, exact determination of the apparent second-order rate constant was not possible (>106m−1 s−1).Elimination of Asp-143 significantly increased Compound I reactivity toward thiocyanate. B. Compoukobs > 0.5 s−1 with 1 mm). Tyrosine did not react at all . Fig. 6C shows the reaction between WT KpDyP Compound I (green spectrum) and 50 μm serotonin at pH 7.0. Compared with the direct two-electron reduction of Compound I to ferric KpDyP, different spectral transitions were observed. During the very slow reaction, a redox intermediate accumulated after 15–20 s with spectral features reminiscent of oxoiron(IV)-type Compound II . Finally, this intermediate slowly disappeared, and the spectrum of ferric WT KpDyP was formed (black spectrum). This conversion (Compound I → Compound II → ferric state) is also reflected by biphasic time traces monitored at 405 and 420 nm but not 386 nm (i.e. isosbestic point between Compound I and Compound II).Next we probed Compound I reduction by serotonin. It has to be noted that the addition of TMB leads to spectral changes comparable with those described for serotonin, whereas ascorbate and hydroquinone mediate only slow spectral conversions (D). Conversion of D143A Compound I to an oxoiron(IV)-type Compound II  was monophasic with clear isosbestic points at 397, 450, 502, and 572 nm. No further transitions were observed, probably due to excess of H2O2 and cycling of the variant. The apparent second-order rate constant kapp of Compound I reduction by serotonin was estimated to be ∼104m−1 s−1.Again, the reactivity of the D143A variant Compound I with serotonin was several orders of magnitude higher at pH 7.0 compared with the WT enzyme D. ConverKpDyP at pH 7.0 and pH 10.0 during the time course of the reaction . Data clearly support the stepwise formation of Compound I and oxoiron(IV)-type Compound II. However, in the WT enzyme (in contrast to the D143A variant), Compound II never fully accumulated. At pH 7.0, always a mixture of dominating Compound I, Compound II (<20%), and ferric state (<10%) is present. This ratio differs at pH 10  . In any case, these findings clearly demonstrate that dye-decolorizing peroxidases of class B follow the classical peroxidase cycle with an oxoiron(IV)-type Compound II as redox intermediate.Finally, we have analyzed the serotonin-mediated reaction of Compound I by the ProK global analysis software (Applied Photophysics). Spectral simulation allowed isolation of the spectral features of ferric state, Compound I, and Compound II of WT KpDyP and the three variants at high resolution. The WT structure was solved by molecular replacement using the DyP structure from V. cholerae (VcDyP). For the variants, the WT structure was used as a replacement model. The structures were refined to a resolution of 1.6 Å (WT), 1.27 Å (D143A), 1.86 Å (R232A), and 1.09 Å (D143A/R232A). Data collection and refinement statistics are given in oF − cF electron density maps contoured at σ = 1.5 of the active site is shown in Fig. S5.Finally, we elucidated the crystal structures of KpDyP. For all figures, we use the chain A subunit, but both subunits are in good agreement with respect to amino acid position, ligands, and intramolecular distances if not noted otherwise. One heme b cofactor is present per subunit, with His-215 acting as proximal ligand at a distance of 2.2 Å between the heme iron and Nϵ. The proximal ligand is hydrogen-bonded to Asp-268.All structures exhibit a dimeric ferredoxin-like fold typical for dye-decolorizing peroxidases, composed of antiparallel β-sheets with four β-strands connected by α-helices. At the distal heme side, a pronounced hydrogen-bonding network is observed, including the proposed catalytic residues Asp-143 and Arg-232, water molecules , and propionate p6. Propionate p6 and p7 are connected by hydrogen bonding via the water molecule W2. Importantly, a salt bridge between Arg-232 and propionate p7 stabilizes the distal architecture. As already observed in other B-class DyP structures, there are two access channels to the heme. The distal access channel to the active-site pocket composed of Asp-143, Arg-232, Phe-248, Leu-246, and Ser-234 is narrow with a calculated bottle neck radius of 1.2 Å . The second access from the surface is via the solvent-exposed propionate p6 .Fig. S5). However, in D143A, the distal access channel is significantly bigger, whereas the surface exposure of propionate p6 remains similar to the WT protein . These structural alterations might cause the different space group of the crystal structure of R232A compared with WT KpDyP despite very similar crystallization conditions  is coordinated by a proximal histidine (His-215), which is hydrogen-bonded with a fully conserved aspartate (Asp-268). The negative standard reduction potential E°′ of the Fe(III)/Fe(II) couple of −350 mV clearly suggests a pronounced imidazolate character of His-215 in KpDyP. In other B-class DyPs, the corresponding E°′ values were reported to be more positive (e.g. −260 mV in PpDyP (ElDyP (E°′(Fe(III)/Fe(II)). However, in D143A, the small effect (+20 mV) seems to reflect the increase in exposure of the heme iron to solvent compared with WT KpDyP (E°′ (−3 mV), which indicates that the loss of Arg-232 is compensated by loss of the negatively charged carboxylate group of Asp-143 that points away from the heme iron in R232A . IntereWT KpDyP . By contin R232A . In any KpDyP is stable between pH 4.5 and pH 10.0, as is reflected by almost identical HS spectra determined by both UV-visible and EPR spectroscopy. No alkaline transition was observed, suggesting identical distances between Asp-143, Arg-232, W1, and the heme iron in this pH regime. The variant D143A is unique in showing an alkaline transition (paK = 8.6) to a HS hydroxo state. For each KpDyP variant, EPR revealed the presence of multiple HS species with varying rhombicity, as reflected by the different E/D values (B). Co-existence of several HS species is not uncommon for peroxidases   as well as the propionate side chains p6 and p7 of WT D values B. Co-exioxidases . Differeependent . The EPRn method , NaCl exn method , pH, andn method . On the ectively , 23. They , monophasic, and pH-independent between pH 4 and pH 9. CW EPR spectroscopic data recorded at 2.5 K revealed the electronic structure of Compound I to be an oxoiron(IV) porphyrin radical species. This is also reflected by the monitored UV-visible spectral changes (i.e. 50% hypochromicity in the Soret region) during the reaction between ferric KpDyP and equimolar H2O2.Oxidation of ferric WT 2O2 with the ferric heme iron (k1)  (k2). Compound 0 formation is followed by rapid heterolytic cleavage of the peroxide bond (k3), thereby forming Compound I and water. In two heme peroxidase superfamilies , a fully conserved distal His–Arg pair supports Compound I formation. The distal His acts as acid–base catalyst and deprotonates the incoming H2O2 and protonates the distal oxygen during heterolytic cleavage. Additionally, the negatively charged outer oxygen atom seems to be transiently stabilized by the guanidinium group of the conserved arginine (7m−1 s−1) (k3 ≫ k2). As a consequence, Compound 0 is difficult to trap.Compound I formation in heme peroxidases requires an initial interaction (“ligand binding”) of Hron (k1) . This isfamilies  (i.e. pearginine . The ratm−1 s−1)  and sligaK of the side chain of Asp-143 is far below 4.0 because its conjugated base is stabilized through the proximity to the guanidinium group of Arg-232. In the absence of Asp-143, the apparent bimolecular rate constant of Compound I formation is reduced by 3 orders of magnitude at pH 7.0, and the pH independence disappears. Interestingly, whereas a similar impact was observed in E. lignolyticus (2m−1 s−1), the same substitution had only a minor impact on Compound I formation in R. jostii (4m−1 s−1 compared with 2 × 105m−1 s−1 in the WT protein). However, in A- and C/D-class DyPs, it was demonstrated that the conserved distal aspartate is essential in Compound I formation , must also be deprotonated before binding to ferric heme proteins. Thus cyanide complex formation mirrors H2O2-mediated formation of Compound 0 in peroxidases. Typically, the apparent bimolecular rate constant of cyanide complex formation of WT KpDyP is pH-independent, whereas cyanide binding to D143A increases by 1 order of magnitude per pH step between pH 5.0 and pH 9.0. Consequently, our data suggest that Compound 0 formation (but not heterolytic cleavage of the O–O bond in Compound 0) is the rate-limiting step in B-class DyPs. This was also proposed for Compound I formation in ElDyP based on studies on solvent isotope and viscosity effects , we cannot provide clear evidence of its role in heterolytic cleavage of hydrogen peroxide. In Compound 0, the hydroperoxyl anion must replace W1   and 102 (kapp ∼3 × m−1 s−1)  , 36. Them−1 s−1) , 38, res unknown .KpDyP is very stable, as reflected by the absence of spectral shifts within minutes. No internal electron transfer reactions occur, which in other heme peroxidases lead to Compound I* formation with typical oxoiron(IV)-type Compound II spectrum (according to Reaction 3) (i.e. −350 mV). Typically, E°′ values of the Fe(III)/Fe(II) couple strictly correlate with E°′ values of the catalytically relevant redox couples Compound I/Compound II as well as Compound I/ferric state in heme peroxidases /Fe(II) is (ranging from −350 to +50 mV)  . Compounoxidases –44. The  +50 mV) , the morKpDyP, the extraordinary stability of Compound I strictly correlates with very poor peroxidase activities. Within physiological (i.e. cytosolic) pH regimes (pH 5.0–8.0), Compound I of this cytosolic peroxidase did not react with two-electron donors like chloride or bromide and to a limited extent with iodide. Only thiocyanate induced a direct but very slow conversion to the ferric resting state (k4). Halides and thiocyanate typically bind at the distal heme side of peroxidases close to the oxoiron(IV) oxygen of Compound I  is typically very fast with all so far studied heme peroxidases from other superfamilies (k5) and Compound II reduction (k6). Interestingly, and similarly to SCN− oxidation (k4), D143A Compound I reduction was several orders of magnitude faster compared with the WT enzyme. Both the WT and D143A crystal structures show an almost identical heme access via the broad channel leading to propionate p6. However, elimination of Asp-143 opens the entrance to the distal cavity, probably facilitating substrate access to the heme moiety. In any case, during turnover of D143A, Compound II accumulated, suggesting that Compound I reduction rates are significantly higher than Compound II reduction rates (k5 > k6) in this variant.Finally, for the first time, we could identify spectroscopically the formation of an oxoiron(IV)-type Compound II in a B-class DyP with typical absorbance maxima at 418, 527, and 553 nm, when Compound I was mixed with serotonin as a one-electron donor. This is in contrast to other reports on redox intermediates of the peroxidase cycle of B-class DyPs . Due to families . The facKpDyP is pH-independent, very fast, and initiated by rate-limiting Compound 0 formation. The fully conserved distal aspartate is responsible for deprotonation of H2O2, whereas arginine 232 is important for maintaining the distal heme cavity architecture and might support heterolysis of the O–O bond by transiently and electrostatically interacting with the negatively charged distal oxygen atom. Compound I, an oxoiron(IV) porphyrin radical species, is very stable and only very slowly reacts with two- and one-electron donors. Reduction of Compound I by thiocyanate leads directly to ferric KpDyP, whereas reduction by serotonin forms oxoiron(IV)-type Compound II as redox intermediate. Elimination of Asp-143 significantly enhances the reactivity of Compound I with both one- and two-electron donors. Based on our data, we challenge the proposal that peroxidase activity toward conventional aromatic substrates or dyes is related to the physiological role of B-class DyPs. At the moment, we are not able to answer the question about in vivo electron donor(s) of Compound I of B-class DyPs. A study on potential cytosolic interaction partners is in progress.In conclusion, heterolytic cleavage of hydrogen peroxide or binding of cyanide by KpDyP was purchased from ATUM  and cloned in frame into pGEX-6P1 using a Gibson Assembly kit  and primers 5′-TCAGTCACGATGCGGCCGCTCGATCAACCGAGCGCTTGAATACGCTC-3′ and 5′-TCCAGGGGCCCCTGGGAATGAGCCAAGTACAGAGCGGT-3′ for protein production using the GSTrap system. The variants were created by site-directed mutagenesis using the QuikChange Lightning kit  according to the manufacturer's description and primers 5′-GTCTGGTTTCGTGGCCGGCACCGAGAACC-3′ and 5′-GGTTCTCGGTGCCGGCCACGAAACCAGAC-3′ for D143A,5′-GGCCTGAAGATTGTGGCCCAGAGCTTGCCGTA-3′ and 5′-TACGGCAAGCTCTGGGCCACAATCTTCAGGCC-3′ for R232A, and a combination of both primer sets for the double variant.The codon-optimized DNA for E. coli BL21 (DE3) carrying the pLysS plasmid  in Luria broth supplemented with ampicillin and chloramphenicol. Cells were incubated at 37 °C and 180 rpm until A600 = 0.8, and then the cultivation temperature was reduced to 16 °C. Protein expression was induced after 1 h using 0.5 mm isopropyl-β-d-thiogalactopyranoside , and after 16 h of growth at 16 °C, cells were harvested by centrifugation  and stored at −30 °C.The recombinant proteins were expressed heterologously in m Tris-HCl, pH 7.3, 150 mm NaCl, 0.1% Triton X-100) with 1 mm phenylmethylsulfonyl fluoride and 100 μm hemin . Cells were lysed by sonication  and clarified by centrifugation . The cleared lysate was filtered  and loaded onto a GSTrap HP 5-ml  column pre-equilibrated with binding buffer . The loaded columns were washed with binding buffer and pre-equilibrated with cleavage buffer . For on-column cleavage, columns were manually loaded with GST-tagged HRV 3C protease diluted in 5 ml of cleavage buffer and incubated overnight at 4 °C. Elution was performed with 5 ml of cleavage buffer. The eluted protein solution was concentrated using 50-ml Amicon centrifugation units (30-kDa cutoff). The concentrated protein was applied to a HiLoad 16/60 Superdex 200 prep grade column , pre-equilibrated with 50 mm phosphate buffer, pH 7.0, and collected in 500-μl fractions. All fractions with a Reinheitszahl value > 1.9 for the WT protein and the D143A variant and 2.5 for the variants R232A and D143A/R232A were collected, concentrated to > 500 μm, aliquoted, and stored at −80 °C.For protein purification, cell pellets were thawed in lysis buffer (50 mKpDyP with a concentration of 10 μm were prepared in 50 mm phosphate buffer (pH 7.0) and used for UV-visible analysis. For all experiments involving a comparison between pH 4.0 and 8.0, 50 mm phosphate citrate buffer was used, and between pH 8.0 and 10.0 50 mm glycine NaOH was used.All UV-visible spectra were measured at room temperature on a Hitachi U-3900 spectrometer. For each measurement, 1000 μl of −1. The instrument was flushed with nitrogen at a flow rate of 5 liters min−1 and allowed simultaneous UV-visible and ECD monitoring. Samples (400 μl of enzyme solution (10 μm) in 5 mm phosphate buffer (pH 7.0) were measured at room temperature. For ECD spectra in the near-UV and visible ranges (260–450 nm), the bandwidth was set to 3 mm, the path length was 10 mm, and the scan speed was 5 s nm−1. Samples consisted of 1 ml of enzyme solution (10 μm) in 50 mm phosphate buffer (pH 7.0).For secondary structure prediction, ECD spectra were taken in the far-UV (180–260 nm) using Chirascan from Applied Photophysics , the bandwidth was set to 3 mm, path length was 1 mm, and scan speed was 10 s nmm protein was prepared in 50 mm phosphate buffer, pH 7.0. In all cases, 30% glycerol was added as a cryoprotectant. Typically, 100 μl was inserted in a quartz EPR tube and flash-frozen in liquid N2. In addition, the WT protein was activated by adding a 5-fold molar excess of H2O2 to generate Compound I. The EPR sample tubes were vacuum-pumped to 1 millibar during the experiments to remove excess of paramagnetic dioxygen. X-band CW EPR experiments were performed on a Bruker ESP300E spectrometer operating at a microwave frequency of ∼9.44 GHz equipped with a liquid-helium cryostat (Oxford Inc.) to enable temperatures from 2.5 K up to room temperature. Calibration of the magnetic field was done using a Bruker ER035M NMR gaussmeter.For EPR spectroscopic studies, 500 μAll spectra of the ferric proteins were recorded at 4 K under nonsaturating conditions at 1-milliwatt microwave power and 0.5-mT modulation amplitude. The detailed EPR spectrum of the organic radical in the resting state protein was recorded at 80 K using 0.1-milliwatt microwave power and 1-mT modulation amplitude. The EPR spectrum of compound I was recorded at 2.5 K using 100-microwatt microwave power and 0.1-mT modulation amplitude. In all cases, the modulation frequency was 100 kHz. Simulation of the experimental spectra was done using the Matlab -based software Easyspin .−1 cell pressure using a VP-capillary DSC microcalorimeter from Microcal (cell volume: 137 μl), equipped with an autosampler for 96-well plates. All samples consisted of 10 μm enzyme in 50 mm phosphate–citrate buffer, pH 3.0–7.5 (WT) and pH 4.5–7.5 (variants) and were kept at 4 °C before measurement. Collected data were corrected for buffer baseline, normalized for protein concentration, and analyzed using the Microcal Origin software package.Temperature-induced pH-dependent protein unfolding studies were performed using DSC. All samples were analyzed between 20 and 100 °C. Samples were heated at 60 psi (4.136 bar) with a rate of 60 °C hE°′, of the Fe(III)/Fe(II) couple was determined using a homemade OTTLE cell  plus 100 mm NaCl. Additionally, 25 μm methyl viologen and 1 μm lumiflavin 3-acetate, methylene blue, phenazine methosulfate, and indigo disulfonate were used as redox mediators.The standard reduction potential, TLE cell , 50–52. lectrode , 50–52. m phosphate–citrate buffer, pH 4.0 to pH 7.0, with 100 μm, 1 mm, or 10 mm substrate, 500 μm H2O2, and 200 nm enzyme. All reactions were carried out in triplicates.Substrate specificity was determined by following the increase or decrease of absorbance at specified wavelengths for 120 s using a stirred cuvette and a scanning photometer . A typical reaction consisted of 1 ml of 50 mKpDyP–cyanide complex, 2 μm ferric protein in 50 mm phosphate buffer, pH 7.0, was mixed with at least a 10-fold excess of cyanide in the same buffer. The reaction was monitored by following the increase of absorbance at 420 nm. For studies on the pH dependence of cyanide binding, 2 μm enzyme in 5 mm phosphate citrate buffer, pH 7.0, was mixed with at least 10-fold excess of cyanide in 100 mm phosphate–citrate buffer, pH 3.0–8.0, or 100 mm glycine-NaOH buffer, pH 8.0–10.0.All transient-state experiments were conducted with a stopped-flow apparatus (SX-18MV or pi-star fitted equipped with diode array detector or a monochromator) from Applied Photophysics. The optical quartz cell with a path length of 10 mm had a volume of 20 μl. The fastest time for mixing was 0.68 ms, and all measurements were performed at 25 °C. For single wavelength measurements, a minimum of three runs were performed for each ligand or substrate concentration. Typically, in studies of the kinetics of formation of the m protein was prepared in 50 mm phosphate buffer, pH 7.0. The titrations were performed in a stirred cuvette at 25 °C and monitored by UV-visible absorption spectroscopy between 250 and 700 nm using a scanning photometer . Cyanide was added stepwise from a 1 m stock solution. All spectra were corrected for the dilution and smoothed using the Savitzky–Golay smoothing algorithm provided by the Hitachi U-3900 software package.In addition to elucidation of the kinetics of cyanide binding to ferric B-class DyP, the thermodynamics of the low-spin complex formation was investigated. In detail, for ligand titration, 1 ml of 5–10 μm ferric enzyme in 50 mm phosphate buffer, pH 7.0, was mixed with at least a 10-fold excess of H2O2 in the same buffer. The pH dependence of this reaction was measured with 2 μm enzyme in 5 mm phosphate citrate buffer, pH 7.0, mixed with at least 10-fold excess of hydrogen peroxide in 100 mm phosphate citrate buffer, pH 3.0–8.0, or 100 mm glycine NaOH buffer, pH 8.0–10.0.For studying the kinetics of Compound I formation, the conventional stopped-flow mode was used. In a typical experiment, 2 μKpDyP and the variant D143A Compound I with the two-electron donor thiocyanate and the one-electron donor serotonin, the sequential stopped-flow mode was used. In detail, Compound I was formed by mixing 2 μm ferric WT KpDyP with a 2-fold stoichiometric excess of hydrogen peroxide in 50 mm phosphate buffer, pH 7.0. After a delay time of 100 ms, Compound I was mixed with varying concentrations of thiocyanate or serotonin. In the case of the D143A variant, Compound I was preformed by mixing 2 μm D143A with a 500-fold excess of H2O2, and the delay time was 3000 ms.For studying the reactivity of WT −1 in 50 mm phosphate buffer, pH 7.0. The seed solution was prepared by collecting all crystals of a single well in 50 μl of 50 mm phosphate buffer, pH 7.0, containing 10 1.0-mm glass beads followed by pulverization through 4 × 1-min vortexing at full speed. The initial seed stock was diluted, depending on the amount of crystals used. The reservoir was filled with 40 μl of crystallization solution. Crystallization drops were set up with enzyme/seed/crystallization solution ratios of 100:100:200, 200:100:200, and 300:100:200 nl. Crystallization plates were stored at 22 °C.All enzymes crystallized in optimized conditions of SG1 Screen HT-96  well C9. Crystallization experiments were performed using SWISSCI MRC three-well crystallization plates (Molecular Dimensions), adopting the vapor diffusion method. Crystallization drops were set using a Mosquito LCP . All protein stocks were prepared in a final concentration of 8.25 mg mlKpDyP crystallized in 23% (w/v) PEG 3350, 0.1 m MgCl2, 0.1 m Tris-HCl, pH 8.5; the D143A variant in 23% (w/v) PEG 3350, 0.1 m MgCl2, 0.1 m Tris-HCl, pH 8.5; R232A in 29% (w/v) PEG 3350, 0.15 m MgCl2, 0.1 m Tris-HCl, pH 8.5; and D143A/R232A in 23% (w/v) PEG 3350, 0.1 m MgCl2, 0.1 m Tris-HCl, pH 8.5, 5% (w/v) glycerol. For cryoprotection, the crystallization conditions were supplemented with 25% (w/v) glycerol. All crystals were harvested using cryoloops and flash-vitrified in liquid nitrogen.WT 5DE0, DyP type B from V. cholerae , isotropic B-factor model, the automated addition of hydrogens and water molecules, optimization of X-ray/ADP weight, and optimization of X-ray/stereochemistry weight. The model was validated with MolProbity 6FKS, 6FL2, 6FKT, and 6FIY.Data sets were collected at beamline ID30A-1  of the E54–cholerae  or the Wing Coot  and maxiing Coot . PHENIX-ing Coot . Final slProbity , 61, andlProbity . Metal cDimensions of putative access channels were calculated using the PyMOL plugin CAVER version 3.0.1. To calculate the access channel, the heme iron position was used as starting coordinates, and heme was excluded. Minimum probe radius was 0.9, shell depth was set to 10, and shell radius was set to 9.V. P., K. N., M. B., H. M., G. M., and S. H. data curation; V. P., K. N., M. B., G. B., S. V. D., P. G. F., and S. H. formal analysis; V. P. validation; V. P. investigation; V. P. visualization; V. P., K. N., S. V. D., and C. O. writing-original draft; V. P., G. B., S. V. D., P. G. F., S. H., and C. O. writing-review and editing; G. B., K. D.-C., S. V. D., and C. O. supervision; S. V. D., P. G. F., and C. O. conceptualization; C. O. funding acquisition.