Here, we describe an acetylation site of Cytc, lysine 39 (K39), which was mapped in ischemic porcine skeletal muscle and removed by sirtuin5 in vitro. Using purified protein and cellular double knockout models, we show that K39 acetylation and acetylmimetic K39Q replacement increases cytochrome c oxidase (COX) activity and ROS scavenging while inhibiting apoptosis via decreased binding to Apaf-1, caspase cleavage and activity, and cardiolipin peroxidase activity. These results are discussed with X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that K39 acetylation is an adaptive response that controls electron transport chain flux, allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.Skeletal muscle is more resilient to ischemia-reperfusion injury than other organs. Tissue specific post-translational modifications of cytochrome  c on K39 acts as a molecular switch in ischemic skeletal muscle, but not other tissues, to increase respiration and prevent apoptosis. This gives skeletal muscle robust resilience to ischemia and ischemia-reperfusion injury.The authors report that acetylation of cytochrome  In the mitochondrial intermembrane space (IMS), it acts as a single electron carrier from complex III to complex IV, also known as cytochrome c oxidase (COX), as part of the electron transport chain (ETC)1. Apart from respiration, Cytc plays a key role in intrinsic apoptosis, where its release from the IMS into the cytosol allows Cytc to bind apoptosis protease activating factor-1 (Apaf-1), activating the apoptosome, which in turn activates caspase-9 and the downstream caspase cascade4. Cytc possesses cardiolipin peroxidase activity, another function related to cell death5. Cardiolipin is a lipid that makes up about 20% of the inner mitochondrial membrane (IMM) lipid composition. During apoptosis, cardiolipin is oxidized by Cytc, which assists in outer membrane permeabilization7. Additionally, Cytc scavenges reactive oxygen species (ROS)9. Given the numerous critical processes carried out by Cytc, the protein is tightly regulated by several mechanisms: allosteric regulation by ATP, expression of tissue-specific isoforms (somatic and testes), and post-translational modifications10.Cytochrome c. Five of the modifications have previously been found to reduce the capacity of Cytc to perform its roles in respiration and apoptosis. The phosphorylations are tyrosine 97 in the heart12, tyrosine 48 in the liver15, threonine 28 and threonine 58 in the kidney18, and serine 47 in the brain20. These phosphorylations were generally found under basal conditions and lost during ischemia, providing a mechanism for reperfusion injury when oxygen is reintroduced into the tissue. Another phosphorylation of Cytc on threonine 49 (numbering based on mature Cytc which lacks the start methionine) was reported to increase in aged mouse hearts21, but, as the authors do not report knocking out endogenous Cytc prior to transfecting in recombinant, phosphomimetic Cytc, data interpretation regarding functional effects of this modification are limited. In prostate cancer, acetylation of lysine 53 promotes two central hallmarks of cancer: switching to Warburg metabolism and evasion of apoptosis23.Six tissue-specific phosphorylation sites and one prostate cancer-specific acetylation site have been characterized on Cytc purified from porcine tibialis anterior (TA) skeletal muscle. Uniquely, K39 acetylation was found after ischemia and was absent under basal conditions. Much like other tissue types, skeletal muscle can experience ischemia-reperfusion injury. Total knee arthroplasty and other conditions that require the application of a tourniquet commonly cause ischemia-reperfusion injury in skeletal muscle25. Other conditions, such as peripheral artery disease or compartment syndrome, also induce ischemic changes in skeletal muscle27. Despite this, skeletal muscle is uniquely resistant to ischemia-reperfusion injury compared to other tissues, such as the brain29. Using purified acetylated and recombinant acetylmimetic protein and a mammalian cell overexpression acetylmimetic system, we studied the effects of this modification on Cytc function. We report that this acetylation stimulates respiration in the ischemic muscle to meet the increased energy demand. Additionally, this modification reduces the pro-apoptotic capabilities of the protein, protecting the skeletal muscle under conditions of stress. In conjunction with X-ray crystallography and nuclear magnetic resonance (NMR) studies of our Cytc variants, we propose that K39 acetylation of Cytc contributes to the resilience of skeletal muscle to ischemia-reperfusion injury.Here, we describe the identification and characterization of an acetylation site on lysine 39 (K39) of skeletal muscle Cytc was purified from 6 control and 6 ischemic TA samples  under conditions that preserve post-translational modifications. Mass spectrometry analysis revealed an acetylation site on K39 of Cytc that was not present in any of the control samples but was detected in 3 of the ischemic TA samples , making this an ischemia-specific acetylation  via the PRIDE30 partner repository, with the dataset identifier PXD040915. Note: In the raw mass spectrometry results, amino acids were numbered including the start methionine (mature Cytc lacks the start methionine), therefore the numbering will be one number higher than what is reported here for the mature protein. Lysine acetylation of Cytc was also identified on K27, K79, and K86 in some samples, however, these acetylations were present in both the control and ischemic TA samples  muscle was harvested and either immediately snap-frozen (control TA) or exposed to ischemic treatment prior to being snap-frozen (ischemic TA). Cytion Fig. . All masc was analyzed by mass spectrometry or western blot  is nonacetylated. To mimic lysine acetylation, K39 was mutated to a glutamine residue (K39Q), which is a polar, uncharged residue like acetyl-lysine. Using glutamine as a mimetic for acetyl-lysine is an established technique for both Cytc23 and other proteins in general due to their similar side chains, which are both uncharged amides34. As another control, K39 was replaced with arginine (K39R), which retains a positive charge at the residue like unacetylated lysine. As an additional experimental control, K39 was mutated to a glutamate residue (K39E), which places a negative charge at the residue. Recombinant WT, K39R, K39Q, and K39E proteins were purified after bacterial overexpression. LabSafe GEL Blue staining of the recombinant proteins resolved on an SDS polyacrylamide gel demonstrates high purity of Cytc  and superoxide (rate of reduction). To determine the rate of oxidation, recombinant ferro-(Fe2+)-Cytc proteins were reacted with 100 µM H2O2, and the initial reaction was monitored spectrophotometrically via the absorbance of the α peak at 550 nm which is present in reduced, ferro-(Fe2+)-Cytc and greatly diminished in the oxidized, ferri-(Fe3+)-Cytc. The rates of oxidation of K39Q and K39E increased by 66% and 63%, respectively, compared to WT -Cytc proteins were reacted with superoxide, and the initial reaction was monitored spectrophotometrically at 550 nm. The rates of reduction of recombinant K39Q and K39E Cytc decreased 94% and 54%, respectively, compared to WT  at 1.36 Å has continuous omit map density for residue 39 in chain-C when contoured at 1.0 root mean square deviation (RMSD) and for all four chains (chains A through D) when contoured at 0.7 RMSD  at 1.5 Å has a lower overall RSCC value of 0.890, but all residues have continuous backbone density at 1.0 RMSD. The fractional occupancies for the four acetyl groups  and K53Q (7LJX)22, tend to more globally perturb the entire Cytc structure compared to phosphomimetic mutations, T28E (5DF5)16 and S47E (6N1O)19, which show much fewer differences compared to native, rodent WT (5C0Z)19  NMR spectra between the recombinant WT protein and the recombinant K39R, K39Q, and K39E proteins were calculated. Resonance assignment for each recombinant protein was confirmed via recording 3D 15N-1H Nuclear Overhauser Effect Spectroscopy (NOESY)-HSQC and 3D 15N-1H TOtal Correlation SpectroscopY (TOCSY)-HSQC spectra  values , though these values are lower than that of the human WT Cytc protein (6.33 ± 0.02 ns)14. However, the K39Q substitution slightly affected both relaxation rates (R1 and R2) parameters  and spin-spin (R2) relaxation rates of the recombinant WT protein revealed a possible conformational exchange in the μs-to-ms timescale. Additional 15N{1H} NOE experiments were recorded with the recombinant K39E and K39R proteins at 500 MHz  cell line was employed as an additional control for all cellular experiments. Clones of each Cytc variant were selected based on similar expression of Cytc . Therefore, mutations to Cytc may impact intact cellular respiration. The basal oxygen consumption rate of the K39Q cell line demonstrated a 70% increase compared to WT . ΔΨm levels of the cell lines were measured using JC-10, a ratiometric probe. Overall, the trend in ΔΨm matched the trend in respiration with EV < WT < K39R < K39Q < K39E. ΔΨm of the K39Q Cytc cell line demonstrated a 42% increase in red/green fluorescence compared to WT, indicating higher ΔΨm  model43, which simulates ischemia-reperfusion injury in cell culture as previously described20.After exposure to stressful conditions, such as reperfusion following ischemia, ΔΨm and mitochondrial ROS levels of the cell lines were measured at normoxia (control) and after OGD/R. For ΔΨm, each cell line showed a significant change in JC-10 red/green fluorescence ratio after exposure to OGD/R compared to the normoxia control. Interestingly, following OGD/R, the K39Q cell line demonstrated an increase of only 4% JC-10 red/green fluorescence compared to the WT cell line, mitigating the larger difference seen when the experiment is run under conditions of normoxia  staining by flow cytometry following treatment with Hath Fig. . Treatmeath Fig. . The higath Fig. . The repath Fig. . Overallc to its negatively charged binding pocket on Apaf-1 is mediated largely by electrostatic interactions3. As discussed above, K39 of Cytc is known to be part of the binding site to Apaf-145. Removing the positive charge at K39 perturbs this interaction, which in turn reduces downstream apoptotic signaling. In line with this, the cell line expressing acetylmimetic K39Q Cytc showed reduced levels of Cytc pulled down after Apaf-1 immunoprecipitation compared to the cell line expressing the WT , which affect the surroundings of the heme group, likely explain the increase in COX activity observed here by facilitating more efficient electron transfer from Cytc to COX. Our previous work on Cytc phosphorylation under basal conditions revealed an inhibition of respiration, maintaining an intermediate, optimal ΔΨm20. This protection was lost during ischemia which results in mitochondrial hyperpolarization upon reperfusion. In this study, we show that K39 acetylation, which is gained during ischemia, follows a different strategy to meet the energy demands of the cell by stimulating respiration.The reaction between Cytm. Maintaining an intermediate, optimal ΔΨm of 80 to 120 mV provides the full capacity to produce ATP while preventing excessive ROS generation52. When mitochondria are hyperpolarized, such as after ischemia-reperfusion, and ΔΨm rises above 140 mV, ROS produced at complexes I and III increases exponentially53. The relationship between respiration, ΔΨm, and ROS is well supported by the data presented here. Under basal conditions, ΔΨm of the K39Q cell line was higher than the WT. Similarly, the K39Q cell line showed higher mitochondrial ROS levels under basal conditions. Using our OGD/R model, we also assessed the effect of our acetylmimetic K39Q stable cell line on ΔΨm and ROS after simulated ischemia-reperfusion. Interestingly, the K39Q stable cell line did not show a further increase in ROS after OGD/R, suggesting that the acetylmimetic replacement already supports higher ΔΨm and ROS levels under basal conditions.Respiration drives the generation of the ΔΨ2O2. The acetylmimetic showed a reduced capability to scavenge superoxide, which is an important signaling molecule in normal muscle contraction56. Therefore, reduced scavenging of superoxide could be an adaptation to short periods of hypoxia that occur during normal muscle exercise. However, this response would become maladaptive in the context of ischemia-reperfusion injury where pathological superoxide levels mediate cellular damage. In contrast, the recombinant K39Q protein demonstrated an increased ability to scavenge H2O2.The purified recombinant K39Q protein demonstrated an interesting relationship with superoxide and Hc is its involvement in apoptosis, where it performs multiple roles. When released from the mitochondrial IMS to the cytosol, Cytc binds Apaf-1, activating the apoptosome and committing the cell to apoptosis. Previous publications strongly suggested that K39 is a part of the Cytc-Apaf-1 binding domain, where it is believed that Cytc K39 forms a hydrogen bond with the carbonyl carbon of Apaf-1 phenylalanine 106345. Our biochemical and cellular analysis of K39Q revealed that the substitution of the lysine with glutamine dramatically reduces the pro-apoptotic capability to activate downstream caspases. Additionally, the recombinant K39Q protein displayed reduced cardiolipin peroxidase activity and the K39Q cell line exhibited less cell death after exposure to H2O2, prolonged OGD/R, or ER stress. Furthermore, pro-apoptotic signaling was blocked in the K39Q cell line. Apaf-1 pull-down resulted in lower levels of K39Q Cytc co-immunoprecipitation, and the K39Q cell line showed lower levels of caspase-9 and caspase-3 cleavage following induction of intrinsic apoptosis. These data further support a protective role for Cytc K39 acetylation in skeletal muscle by reducing the ability of the protein to activate apoptosis.Another major function of Cyt58. In this publication, Cytc, located in the IMS, was found to gain acetylation on K39 in tibialis anterior skeletal muscle as a response to ischemia. Porcine Cytc has 18 lysines, which makes K39 acetylation a hypoxia-selective modification. This specificity adds to the likelihood that a specific acetyltransferase mediates Cytc K39 acetylation. Interestingly, deletion of the acetyltransferase GCN5L1 in cardiomyocytes renders cells more sensitive to ischemia-reperfusion injury, with larger infarcts in the knockout hearts59, and a previous publication found enrichment of GCN5L1 in the intermembrane space60. A second acetyltransferase candidate, ACAT1, was co-fractionated with Cytc in a previous publication61. Elucidation of the specific acetyltransferase involved in the signaling pathway for this acetylation will be necessary to fully understand the role of this modification. Interestingly, we found that K39 acetylation was removed by sirtuin5 in vitro. Sirtuin5 has previously been shown to co-localize to the IMS with Cytc62, and Cytc was previously suggested to be a substrate of sirtuin5-mediated deacetylation on an unknown site63. Noteworthy, sirtuins have been reported to be downregulated as a response to hypoxia64, suggesting that blocking the removal of Cytc K39 acetylation during ischemia facilitates the protective effect seen in skeletal muscle.Lysine acetylation is the most common post-translational modification in the mitochondria, particularly in the matrix where the basic pH and high concentrations of acetyl-CoA promote non-enzymatic acetylation65. It is known that the median stoichiometry for lysine acetylation, in general, is 0.02%, with highly acetylated proteins such as histones and acetyltransferase proteins being the only proteins to ever approach or even exceed 1% acetylation69. Despite this, lysine acetylation is known to be a highly biologically relevant modification70. By these standards, our results demonstrate that the ischemia-induced K39 acetylation of Cytc is a more abundant acetylation, as detailed in Supplementary Table c at 1.50 Å resolution showed an average occupancy of 75% for the acetyl group, suggesting that a large proportion of the protein in the crystal structure is acetylated. Potential explanations for this discrepancy between the mass spectrometry and the crystallography occupancies may be that K39 acetylated Cytc preferably crystallized within the ischemic TA sample, resulting in an overrepresentation of the acetyl group in the crystallography occupancy, or that some of the modification was lost during the sample preparation for mass spectrometry.Previous work on quantitating lysine acetylation using mass spectrometry indicates that lysine acetylation occurs at very low stoichiometries28. Skeletal muscle is a unique tissue type with drastic changes in perfusion depending on usage72. Cytc K39 acetylation may be a helpful modification during exercise, allowing the muscle to meet the high energy demand while protecting the muscle from cell death. It is possible that this acetylation is a transient modification meant to handle short-term energy deficiencies. Additionally, individual muscles may have unique responses to ischemia. More research must be done to fully elucidate the signaling pathway controlling this acetylation. Identification of the pathway could aid in the development of therapeutic interventions for muscle ischemia-reperfusion injuries, such as during total knee arthroplasty, or chronic ischemic conditions, such as peripheral artery disease.Overall, our findings support the assertion that K39 acetylation is protective during ischemia-reperfusion by enhancing energy production and at the same time disabling apoptotic capabilities. This acetylation may help explain the resilience of skeletal muscle to ischemia compared to more sensitive tissues like the brain19.Porcine (domestic farm swine) tibialis anterior (TA) muscle samples from six animals  were sourced from Wayne State University School of Medicine  as discarded tissues with institutional animal care and use committee approval under protocol IACUC-18-11-0859. Tissues were rapidly harvested from animals post-euthanasia. For each animal, TA muscle from one hindlimb was immediately snap-frozen (control TA), while TA muscle from the other hindlimb was placed in a tightly sealed bag and incubated in a water bath at 37 °C for 1 h to induce ex vivo ischemia prior to being snap-frozen (ischemic TA), as we have previously done to induce ex vivo ischemia in brain tissuec was purified from non-ischemic control and ischemic TA tissue samples following our established protocol used on other tissues and organs in the past19 which preserves post-translational modifications with minor adjustments. After homogenization, the tissue samples were subjected to acid extraction using 100 mM phosphate buffer, pH 3.9 at 4 °C. The pH of the mixture was adjusted to 4.0 using glacial acetic acid. After 10 min, the pH was adjusted to 4.3 using KOH and Cytc was extracted overnight at 4 °C. The next day, samples underwent centrifugation at 24,000 x g for 45 min at 4 °C. The pH of the supernatants was adjusted to 6.5 and protease and phosphatase inhibitors were added to final concentrations of 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium vanadate, and 10 mM KF. The sample was then adjusted to pH 7.4 and underwent centrifugation as above. The conductivity of the resulting supernatants was adjusted to 3.0 mS/cm using ddH2O and the supernatants were loaded onto a DE52-cellulose anion exchange column  equilibrated with 20 mM phosphate buffer, pH 7.4, with a conductivity of 3.0 mS/cm. The flowthrough of the column, which contains Cytc, was adjusted to a conductivity of 5.0 mS/cm using 1 M phosphate buffer, pH 6.5, and to a pH of 6.5 using glacial acetic acid and loaded onto a CM52-cellulose cation exchange column (Whatman) equilibrated with 30 mM phosphate buffer, pH 6.5, and a conductivity of 5.0 mS/cm. The Cytc bound on the CM52 column was oxidized using 2 mM potassium ferricyanide. The Cytc fractions were then eluted using gradient elution from 50 mM to 150 mM phosphate buffer, pH 6.5, concentrated, and further purified via size exclusion chromatography using a Sephacryl S-100 gel filtration column . Purified Cytc samples were buffer exchanged to 20 mM ammonium bicarbonate, pH 7.6, using Amicon Ultra-15 10 kDa centrifugal filter units .Cytc oxidase (COX) was purified from porcine heart following our established protocol73 which preserves post-translational modifications. Briefly, the porcine heart was homogenized, and the tissue extract was loaded onto a DEAE-Sepharose anion exchange column  equilibrated with 125 mM phosphate buffer and 0.5% Triton X-100, pH 7.4. The bound enzyme was eluted from the column using a salt gradient going from 100 mM to 700 mM phosphate buffer with 0.1% Triton X-100, pH 7.4. The enzyme was further purified via ammonium sulfate precipitation.Cytochrome c (40 μg) was analyzed by mass spectrometry at the Max Planck Institute for Molecular Genetics . Samples were initially processed by solubilization in 200 µL of 100 mM ammonium bicarbonate, pH 8, and boiled at 95 °C for 10 min on a rocking platform. Half of each sample was further processed via cysteine reduction by adding 5.5 mM tris(2-carboxyethyl)phosphine at 37 °C for 30 min at 800 rpm and via alkylation by adding 24 mM 2-chloroacetamide at room temperature for 30 min Each sample (pH 8.0) was split into two equal amounts and either digested by 200 ng trypsin or 200 ng chymotrypsin with shaking at 700 rpm at 37 °C for tryptic samples and 25 °C for chymotryptic samples overnight. To enhance enzyme activity, 4.5 μL of 100% acetonitrile was added to the tryptic samples. Peptide desalting was performed using Pierce C18 Spin Tips & Columns  according to the manufacturer’s instructions. Desalted peptides were reconstituted in 5% acetonitrile and 2% formic acid in the water, briefly vortexed, and sonicated in a water bath for 5 min before injection to nano-LC-MS. Each run used 5 µg of digested protein.Purified Cyt74. Briefly, the LC separation was performed using a PicoFrit analytical column  in-house packed with 3 µm C18 resin . Peptides were eluted using a gradient from 3.8 to 38% solvent B  in solvent A (0.1 % formic acid) over 120 min at 266 nL/min flow rate. Nano-electrospray was generated by applying 3.5 kV. A cycle of one full Fourier transformation scan mass spectrum  target 1 × 106) was followed by 12 data-dependent MS/MS scans  with a normalized collision energy of 25 eV. To avoid repeated sequencing of the same peptides, a dynamic exclusion window of 30 sec was used. In addition, only peptide charge states between two to eight were sequenced.LC-MS/MS was carried out by nanoflow reverse phase liquid chromatography  coupled online to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific), as previously describedSus domesticus proteome database UniProtKB with the ID UP000008227 (December 2016)75. Parameters of MaxQuant database searching were a false discovery rate (FDR) of 0.01 for proteins and peptides, a minimum peptide length of seven amino acids, a first search mass tolerance for peptides of 20 ppm, and a main search tolerance of 4.5 ppm. A maximum of two missed cleavages was allowed. Cysteine carbamidomethylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. Post-translational modifications of interest were lysine acetylation, mono-, di-, and tri-lysine methylation, as well as serine, threonine, and tyrosine phosphorylation, which were assessed via independent searches. All lysine acetylations of Cytc were manually verified by the presence of at least one diagnostic peak (m/z 126 or 148 Da)76. The mass spectrometry data have been deposited to the ProteomeXchange Consortium  via the PRIDE30 partner repository with the dataset identifier PXD040915. Note: In the raw mass spectrometry results, lysines were numbered including the start methionine (mature Cytc lacks the start methionine), therefore the numbering will be one higher than what is reported here in the final manuscript text.Raw MS data were processed with MaxQuant software (v2.2.0.0)  and searched against the c of control and ischemic samples were run on a 10% tris-tricine SDS-PAGE gel, transferred onto immuno-blot PVDF membrane  via wet transfer , and blocked in blocking reagent (5% non-fat dry milk in 1x TBS-T with 0.1% Tween 20) for 1 h at room temperature. For acetyl-lysine detection, the membrane was incubated with a 1:800 dilution of rabbit anti-acetyl-lysine conjugated to horseradish peroxidase secondary antibody  in blocking reagent overnight at 4 °C. On a separate gel, one µg of the same samples were run, transferred, and blocked under identical conditions. This membrane was incubated with a 1:4000 dilution of mouse anti-Cytc antibody  in blocking reagent overnight at 4 °C. The next day, the membrane for Cytc was incubated with a 1:8000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody  in blocking reagent for 2 h at room temperature. Blots were visualized using Pierce ECL western blotting substrate .Protein concentration was determined using the DC protein assay kit  according to the manufacturer’s protocol. One µg of purified porcine TA muscle Cytc expression levels of cell lines stably expressing WT, K39R, K39Q, K39E, and negative control empty vector (EV) were assessed via western blot. Cells were lysed using 100 µL RIPA lysis buffer ) supplemented with protease inhibitor cocktail , sonicated, and centrifuged at 16,900 x g for 20 min at 4 °C to remove cell debris. Sixty µg cell lysates were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked as described above. The membrane was cut in half between the 25 and 35 kDa markers. The lower and upper membrane halves were incubated with a 1:1000 dilution of mouse anti-Cytc antibody or 1:5000 dilution of mouse anti-GAPDH antibody  as a loading control, respectively, in blocking reagent overnight at 4 °C. The next day, membrane halves were incubated with a 1:5000 or 1:10,000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody in blocking reagent for 2 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate.Cytg for 20 min at 4 °C to remove cell debris. Sixty µg cell lysates were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked as described above. Membranes were probed using a 1:500 dilution of rabbit anti-PGC-1 antibody , a 1:1000 dilution of rabbit anti-citrate synthase antibody , a 1:2000 dilution of rabbit anti-tubulin antibody  as a loading control, and a 1:1000 dilution of mouse anti-TFAM antibody  overnight at 4 °C. The next day, the membranes were incubated with a 1:2500, 1:1000, or 1:2000 dilution of donkey anti-rabbit IgG conjugated to horseradish peroxidase secondary antibody , respectively, or a 1:5000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody for 1 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate.Cell lines stably expressing WT, K39R, K39Q, K39E, and EV were assessed for PGC-1α, citrate synthase, and TFAM via western blot. Cells were lysed using 100 µL RIPA lysis buffer supplemented with protease inhibitor cocktail, sonicated, and centrifuged at 16,900 x g for 20 min at 4 °C. Five mg cell lysates were incubated with mouse anti-Cytc at 1 μg per mg of sample overnight at 4 °C. The next day, the mixtures were then incubated with Protein A/G PLUS-Agarose beads  overnight at 4 °C. Beads were washed and 40 µL of eluate from the beads were run on a 10% tris-tricine SDS-PAGE gel and transferred as described above. The membrane was blocked in BSA blocking reagent (5% BSA in 1x TBS-T with 0.1% Tween 20) for 1 h at room temperature. The membrane was incubated with a 1:1000 dilution of rabbit anti-acetyl-lysine conjugated to horseradish peroxidase antibody as described above in BSA blocking reagent overnight at 4 °C. On a separate gel, the eluate from the beads were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked under identical conditions. As a loading control, the membrane was incubated with a 1:3000 dilution of mouse anti-Cytc antibody in blocking reagent overnight at 4 °C. The next day, the membrane was incubated with a 1:8000 of dilution sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody in blocking reagent for 1 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate.For the TA muscle acetylation time course, TA muscle samples were snap-frozen (0 min ischemia) or were placed in a tightly sealed bag and incubated in a water bath at 37 °C for 15, 30, 45, or 60 min to induce ex vivo ischemia prior to being snap-frozen. Samples were lysed in IP buffer  supplemented with protease inhibitor cocktail. Additionally, the lysis buffer was also supplemented with 1 mM Trichostatin A  and 5 mM nicotinamide  in order to inhibit acetylase and deacetylase enzymes. Lysed samples were sonicated, incubated for 30 min at 4 °C, and centrifuged at 16,900 x c co-IP, Cytc double knockout mouse embryonic fibroblasts stably expressing WT, K39R, K39Q, K39E Cytc were utilized to assess the interaction between Apaf-1 and Cytc. Cells (3 x 106) were seeded onto a 15 cm cell culture dish and cultured overnight. The next day, cells were washed twice with cold 1x PBS, harvested via scraping, and pelleted via centrifugation. The cell pellets were resuspended in 300 µL binding buffer , lysed via sonication, and centrifuged at 16,900 x g for 20 min at 4 °C to remove cell debris. The supernatants containing Apaf-1 and Cytc were incubated with 1 mM ATP for 30 min at 37 °C. For co-IP, 2 mg total protein from the above solutions were incubated with 4 µg rabbit anti-Apaf-1 antibody  overnight at 4 °C. The next day, the mixtures were then incubated with Protein A/G PLUS-Agarose beads overnight at 4 °C. Beads were washed and 40 µL of eluate from the beads were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked as described above. The membrane was cut in half between the 35 and 40 kDa markers. The lower and upper membrane halves were incubated with a 1:4000 dilution of mouse anti-Cytc antibody or a 1:1000 dilution of rabbit anti-Apaf-1 antibody as a loading control, respectively, in blocking reagent overnight at 4 °C. The next day, membrane halves were incubated with a 1:8000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody or a 1:8000 dilution of donkey anti-rabbit IgG conjugated to horseradish peroxidase secondary antibody, respectively, in blocking reagent for 1 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate.For the Apaf-1 Cytc from purified porcine TA muscle Cytc of control and ischemic samples was performed in 50 mM Tris, pH 8.0, 4 mM MgCl2, and 0.2 mM DTT at 37 °C for 3 h in the presence and absence of 1 mM NAD+ and 200 ng sirtuin5 . The reaction was stopped via denaturation at 95 °C for 5 min. One and a half µg of each sample were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked as described above. The membrane was probed using a 1:1000 dilution of rabbit anti-acetyl-lysine conjugated to horseradish peroxidase antibody as described above. On a separate gel, 40 µL of eluate from the beads were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked under identical conditions. As a loading control, the membrane was probed using a 1:3000 dilution of mouse anti-Cytc antibody followed by a 1:8000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody as described above. The blots were visualized using Pierce ECL western blotting substrate.Three μg of Cytc (WT) cDNA cloned into a bacterial pLW01 expression vector77 was used to generate control K39R, acetylmimetic K39Q, and an additional control K39E Cytc using the QuickChange Lightning site-directed mutagenesis kit  according to the manufacturer’s protocol. The pLW01 plasmid also possesses the sequence for heme lyase, the enzyme necessary to covalently link heme and Cytc, which bacteria lack. The following mutagenesis primers were used in amplification of each Cytc plasmid: K39R forward primer: 5′-CTGTTTGGGCGGAGGACAGGCCAGGC−3′, K39R reverse primer: 5′-AGCCTGGCCTGTCCTCCGCCCAAACAG−3′, K39Q forward primer: 5′-TCTGTTTGGGCGGCAGACAGGCCAGGC−3′, K39Q reverse primer: 5′-GCCTGGCCTGTCTGCCGCCCAAACAGA−3′, K39E forward primer: 5′-TCTGTTTGGGCGGGAGACAGGCCAGGC-3′, K39E reverse primer: 5′-GCCTGGCCTGTCTCCCGCCAAACAGA-3′. The PCR products were incubated for 1 h at 37 °C with DpnI endonuclease to remove methylated parental DNA and then transformed into XL 10-Gold Ultracompetent cells. Plasmids were purified from individual colonies using the Wizard Plus SV miniprep purification system . Purity and quantity were determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). The presence of the desired mutation in each plasmid was confirmed using DNA sequencing .Somatic rodent Cytc constructs were transformed via heat shock into competent E. coli C41 (DE3) cells  for protein overexpression. Transformed bacteria were cultured overnight at 37 °C in Terrific Broth medium  supplemented with 0.1 mg/mL carbenicillin. Once an optical density (OD)600 of 0.8 was reached, Cytc overexpression was induced by the addition of 100 µM isopropyl-β-D-thiogalactoside. After 6 h induction, bacterial cells were harvested and lysed, and Cytc was purified as previously described16. Briefly, cells were pelleted via centrifugation at 8400 x g for 45 min at 4 °C, resuspended in 20 mM phosphate buffer, pH 7.4, supplemented with protease cocktail inhibitor, and then lysed via SLM Aminco French pressure system . Cell debris was removed via centrifugation at 26,200 x g for 45 min at 4 °C. Cytc variants were purified via ion exchange chromatography as described above. Protein concentration was determined spectrophotometrically, while protein purity was determined via LabSafe GEL Blue  staining of a 10% tris-tricine SDS-PAGE gel loaded with 500 ng of each Cytc variant and ran as described above.WT and mutant Cytc was either fully oxidized with several grains of potassium ferricyanide (K3Fe(CN)6) or fully reduced with several grains of sodium dithionite (Na2S2O4). Oxidized or reduced Cytc proteins were run through NAP5 column  to separate the protein from the oxidizing or reducing agent. The concentration of Cytc was calculated using the absorbance at 550 nm of the oxidized and reduced spectra via the following formula: [Cytc] in mM = (Abs550reduced – Abs550oxidized) / (19.6 mM/cm x 1 cm) x dilution factor.The oxidized and reduced spectra of recombinant WT, K39R, K39Q, and K39E proteins were measured from 700 nm to 250 nm using a Jasco V-570 double beam spectrophotometer . Additional oxidized spectra were taken from 750 nm to 620 nm to detect the characteristic heme peak at 695 nm. Cytc were measured using purified regulatory-competent porcine heart COX. This is the same isozyme present in skeletal muscle. COX was diluted to 3 μM in solubilization buffer  supplemented with a 40-fold molar excess of tetraoleyl-cardiolipin  and 0.2 mM ATP. COX was dialyzed in 1 L solubilization buffer using a 12–14 kD Spectra/Por 2 dialysis membrane  overnight at 4 °C to remove bound cholate16. The activity of COX (26.7 nM) in 220 μL solubilization buffer with recombinant Cytc variants (0 – 25 μM) or control and ischemic porcine TA muscle Cytc (5 μM) was measured using a Clark-type oxygen electrode  at 25 °C with 20 mM ascorbate as the electron donor. Oxygen consumption was recorded and analyzed using Oxytrace+ v1.0.48 software (Hansatech). The COX activity is reported as turnover number (s−1).COX activities with the recombinant WT, K39R, K39Q, and K39E and the control and ischemic porcine TA muscle Cytc were measured using rhodamine fluorescence after caspase-3 mediated cleavage of Z-DEVD-R110 substrate. Cytc-/- embryonic fibroblasts  were cultured and lysed for isolation of cytosolic extracts as previously described17. Briefly, the knockout cells were cultured in 8 X 150-mm plates, trypsinized, pelleted, and washed twice with 1X PBS. Next, the pellets were washed once with a cold, hypotonic cytosolic extraction buffer . The cell pellet was resuspended in 1 mL CEB and allowed to swell in the hypotonic CEB for 15 min at 4 °C. The suspension was transferred to a 2 mL Dounce homogenizer, and cells were disrupted via 30 to 35 strokes of the glass pestle. Cell breakage was monitored periodically by observing the homogenate on slides under a microscope. Lysates underwent centrifugation of 15,000 x g for 15 min at 4 °C to remove organelles and nuclei. The cytosolic extract was diluted to 2 mg/mL and incubated with 20 µg/mL of Cytc for 2.5 h at 37 °C. The preincubated Cytc-cytosolic extracts mixtures were utilized with the EnzChek Caspase-3 assay kit  according to the manufacturer’s protocol with the caspase-3 inhibitor, Ac-DEVD-CHO, as a negative control. The reaction was initiated with the addition of the caspase-3 substrate, Z-DEVD-R110, a rhodamine-based tetrapeptide that fluoresces upon cleavage by caspase-3. Rhodamine fluorescence was measured using a Fluoroskan Ascent microplate reader  with excitation/emission wavelengths of 485nm/527 nm excitation as previously described17. Caspase-3 activity is reported as a percentage of change in fluorescence (arbitrary units) compared to WT (recombinant Cytc variant proteins) or control porcine TA muscle Cytc.The downstream caspase-3 activities after activation of Apaf-1 by recombinant WT, K39R, K39Q, and K39E and the control and ischemic porcine TA muscle Cyt2O2 were spectrophotometrically recorded to determine the rate of Cytc oxidation by H2O2 as described78. Cytc variants were fully reduced with Na2S2O4 and subsequently desalted by passing through NAP5 columns, as described above. A cuvette was prepared containing 15 μM ferro-Cytc and 0.2 M Tris-Cl, pH 7.0, and the baseline 550 nm absorbance was measured. An addition of 100 μM H2O2 initiated the reaction. The 550 nm absorbance was measured every 10 s for 2 min The decrease in the 550 nm absorbance corresponds to the oxidation of Cytc. The initial rate of oxidation is reported in μM/s.The kinetics of the redox reaction between WT, K39R, K39Q, and K39E and Hc and superoxide were analyzed spectrophotometrically using a hypoxanthine/xanthine oxidase reaction system80 to determine the rate of superoxide scavenging by Cytc. Cytc variants were fully oxidized with K3Fe(CN)6 and subsequently desalted by passing through NAP5 columns, as described above. A cuvette was prepared containing 10 µM ferri-Cytc, 100 µM hypoxanthine, and 14.2 nM catalase (to prevent H2O2 accumulation) in 1x PBS. The production of superoxide was initiated with the addition of 181.5 nM xanthine oxidase. An addition of 925 nM superoxide dismutase 2 was also added for some experiments as a negative control that rapidly converts superoxide into H2O2. The 550 nm absorbance was remeasured every 15 s for 3 min The increase in the 550 nm absorbance corresponds to the amount of superoxide scavenged by Cytc. The initial rate of reduction is reported in µM/s.The kinetics of the redox reaction between WT, K39R, K39Q, and K39E CytE°’) of WT, K39R, K39Q, and K39E Cytc variants were determined spectrophotometrically via the equilibration with a standard method39 as previously described17. The reference compound was 2,6-dichloroindophenol , which has an absorption peak at 600 nm in the oxidized form. Briefly, 52.5 µM Cytc, 15.87 μM K3Fe(CN)6, 31.75 mM citrate buffer, pH 6.5, and 31.75 μM DCIP were combined and baseline absorbances corresponding to fully oxidized Cytc (Abs550 – Abs570) and DCIP (Abs600) were recorded. Sequential additions of 1 μL of 5 mM ascorbate were added to reduce Cytc, and the Abs550, Abs570, and Abs600 were recorded after each addition in one-minute intervals. At the end of the titration, a few grains of Na2S2O4 were added to fully reduce Cytc and DCIP. In order to calculate E°’, log (DCIPox/DCIPred) was plotted against log (Cytcox/Cytcred). This yielded a linear function with a slope, nDCIP/ncCyt, and a y-intercept, ncCyt/59.2(EcCyt-EDCIP). Using the Nernst equation, these values were used to calculate E°’ for each of the recombinant Cytc variants. E°’ is reported as mV.The midpoint redox potentials 6 and desalted using NAP5 columns, as described above. A cuvette was prepared containing 5 µM ferri-Cytc in 50 mM phosphate buffer, pH 6.1, and the baseline 408 nm absorbance was measured. The degradation of heme was initiated with the addition of 3 mM H2O2. The 408 nm absorbance was measured at 60, 200, 400, 600, and 800 s. The decrease in the characteristic heme Soret band at 408 nm indicates heme degradation. The heme degradation is reported as a percentage of change in absorbance compared to the baseline absorbance.The structural integrity of the covalently attached heme groups of WT, K39R, K39Q, and K39E Cytc were measured via resorufin formation, the oxidation product of Amplex red, as previously described77. Liposomes composed of 0%, 20%, 30%, and 50% of 18:1 tetraoleoyl-cardiolipin  with the remainder composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine  were generated. Lyophilized TOCL and lyophilized DOPC were solubilized separately in chloroform to prepare working stock solutions of 10 mg/mL. The required volumes of the solubilized TOCL and DOPC to create different liposome compositions were mixed. The lipid mixtures were evaporated by blowing nitrogen gas on the liquid, resuspended in 20 mM K-HEPES, pH 7.2, and constituted into liposomes via sonication on ice for 5 x 30 s with 1 min intervals. Liposomes (25 µM) were incubated with 1 µM of the recombinant Cytc variant proteins at room temperature in a Costar 96-well plate  for 10 min to allow for equilibration of Cytc-cardiolipin binding. Baseline resorufin fluorescence was measured using a Fluoroskan Ascent microplate reader with excitation/emission wavelengths of 530 nm/590 nm. The oxidation of cardiolipin was initiated with the addition of 10 µM Amplex red  and 5 µM H2O2. The fluorescence was remeasured at 60 and 300 s, during which the reaction rate was linear. Cardiolipin peroxidase activity is reported as fluorescence/minute (arbitrary units/min).The cardiolipin peroxidase activities of WT, K39R, K39Q, and K39E Cytc and ischemic porcine TA muscle Cytc were buffers exchanged to water using Amicon Ultra-15 10 kDa centrifugal filter units . Samples were oxidized using 5 mM K3Fe(CN)6 immediately prior to crystallization. The approximate protein concentration for crystallization was 10-15 mg/mL for K39Q Cytc and 5-8 mg/mL for porcine K39 acetylated Cytc. The sitting drop method was used with a 1 μL drop size protein plus 1 μL of reservoir solution. The best diffracting crystals of K39Q Cytc and ischemic porcine TA muscle Cytc grew to 0.2 mm x 1 mm size in Wizard Cryo2 screen #30 and 0.2 to 0.3 mm x 1.5 mm size in JBS screen #3A2, respectively (Supplementary Table 8DZL) or following cryoprotection with 20% v/v ethylene glycol (porcine K39 acetylated Cytc: 8DVX). Crystal diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-D at the Advanced Photon Source, Argonne National Laboratory using a Dectris Eiger X 9M detector and integrated using the autoPROC program81. Both structures in Supplementary Table 82. Both structures were also checked multiple times with PDB-REDO83. The refinement process was terminated when the two program suites converged to similar values of R-free. The fractional occupancies of the seven K3Fe(CN)6 molecules in 8DVX  were superposed onto molecule A in the rat WT structure (5C0Za) using YASARA version 20-07-0484. The five superposed structures were then loaded into Molecule Operating Environment 85 and all residues with at least one atom within 3.5 Å of Gln39 in molecule A (8DZLa) were displayed. The comparable figure for Aly39 (8DVX) was prepared using the same protocol.The four molecules in the crystal structure of K39Q (c (5C0Za)19, were calculated for molecule A in the crystal structures of T28E (5DF5a)16, K39Q (8DZLa), S47E (6N1Oa)19, and K53Q (7LJXa)22, using YASARA version 20-07-0484 . The RMSD plot for the backbone atoms was generated with Microsoft Excel using data imported from YASARA.The RMSD values, relative to molecule A in the crystal structure of WT rodent Cytc  as previously described23. All reduced  structural models were built using the WT protein as a template with UCSF Chimera 1.15 software 86. The force field parameters for the reduced heme group87 were used to generate the topology and coordinate files, required for the simulation, with the TLEAP module of AmberTools 2021 88. Each structure was summited to 1 μs MD simulation prior to the calculation of the electrostatic surface potential with UCSF Chimera 1.15 software .The structures used to calculate the electrostatic surface potential were based on the crystallographic structure of mouse CytE. coli C41 (DE3) cells were transformed with WT, K39R, K39Q, and K39E mutant pLW01-Cytc constructs and were plated on LB-agar plates containing 0.1 mg/mL ampicillin. Single colonies were incubated at 37 °C with 150 rpm agitation for 16 h. For the NMR experiments, the overnight culture was transferred to 1 L of minimal M9 medium supplemented with 16.7 μg/mL δ-aminolevulinic acid hydrochloride and 15NH4Cl as the sole nitrogen source. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside at an OD600 of 0.8, and the cultures were incubated for 20 h to allow for protein expression. Cells were harvested by centrifugation at 9000 x g for 10 min at 4 °C, suspended in lysis buffer ), and physically ruptured by sonication. Cellular debris was separated by centrifugation at 14,000 x g for 30 min at 4 °C. Protein purification was carried out by ion chromatography with a Nuvia-S column  using a Fast Protein Liquid Chromatography (FPLC) system (Bio-Rad). Protein purity and sample homogeneity were determined spectrophotometrically and by running 5 µg of each protein sample on a 15% SDS-PAGE, followed by Coomassie blue staining. Dynamic light scattering and tryptic digestion analyses confirmed the monomeric state of the protein samples and the point mutations of the Cytc species.15N-labelled Cytc in 10 mM sodium phosphate buffer, pH 6.5, 5% D2O. Sodium ascorbate was used to ensure the redox state of the sample. Backbone amide group resonances of the recombinant Cytc proteins were monitored by recording 2D 15N-1H Heteronuclear Single Quantum Correlation (HSQC) experiments as previously described23. In order to assign the backbone resonances, additional 3D 15N-1H TOtal Correlation SpectroscopY (TOCSY)-HSQC and 3D 15N-1H Nuclear Overhauser Effect Spectroscopy (NOESY)-HSQC experiments were performed. The experimental data were processed using Bruker TopSpin NMR 4.1.1 software (Bruker), and assignment and chemical shift perturbations (CSPs) were analyzed with NMRFAM-Sparky 1.470 NMR assignment tool 89. The average difference  in 15N relaxation parameters , transversal relaxation rate (R2), and heteronuclear 15N{1H} (NOE)) were obtained from experiments recorded at 500 MHz 1H frequency at 25 °C for the recombinant WT and K39Q proteins. Each difference was calculated from a set of relaxation parameters measured on two biologically independent samples for WT and K39Q Cytc. All attempts at replication were successful. The R1 parameter was calculated using 7 delays ranging from 10 to 1000 ms, and the R2 parameter was calculated using 7 delays ranging from 16.96 to 118.72 ms. The 15N{1H} NOE was determined by recording spectra with and without a 4.2 s long proton saturation period. The spectra were processed using NMRPipe 10.9 and analyzed with NMRDraw 10.990. Peak intensities were extracted from each spectrum, with R1 and R2 parameteres for each protein sample obtained by fitting the data to an exponential decay function: 0 represents the peak intensity at time 0 and It represents the intensity at time t. The R2/R1 ratio was used to estimate the rotational correlation time (τc) of the protein variants using TENSOR 2.0 and ROTDIF 1.1 software92.Protein samples were dialyzed against 10 mM sodium phosphate buffer, pH 6.5, for NMR experiments, which were performed on a 700 MHz Bruker Avance-III  and 500 MHz Bruker Avance-III (Bruker) spectrometers equipped with a cryoprobe. Samples contained 800 µM of reduced c (WT) cDNA was cloned into the pBABE-puromycin expression plasmid  between the BamHI and EcoRI restriction sites with the following outer primers: forward primer 5’-CATGGGTGATGTTGAAAAAGGCGAGAAGATTTTTGTTCAAAAGTG−3’ and reverse primer 5’-CACTTTTGAACAAAAATCTTCTCGCCTTTTTCAACATCACCCATG−3’16. A similar PCR mutagenesis approach and mutagenesis primers as above were used to generate WT, K39R, K39Q, K39E, and negative control empty vector (EV) constructs, which were then transfected into Cytc double knockout mouse embryonic lung fibroblast cells41  using Transfast transfection reagent . The transfected cells were cultured in growth media , 10% FBS , 100 µg/mL primocin , 1 mM sodium pyruvate , 50 µg/mL uridine ) at 37 °C with 5% CO2, unless otherwise stated. Stable cell lines expressing the four recombinant Cytc variants and EV were selected in the presence of 2 µg/mL puromycin.Somatic rodent Cytc, and EV were measured. Cells were seeded at 30,000 cells/well onto a 0.1% gelatin coated Costar 24-well plate  and cultured in 2 mL/well growth media. Every 24 h, cells were washed with 1x PBS and collected via trypsinization. Collected cells were counted using a Moxi Z mini automated cell counter .The growth rate of the stable cell lines expressing WT, K39R, K39Q, K39E Cytc, and EV were measured. Cells (1 x 106) were seeded onto a 10 cm cell culture dish  and cultured overnight. The next day, in under 90 s, the cells were washed once with 1x PBS, harvested via scraping, pelleted via centrifugation, and flash frozen in liquid nitrogen. The pellets were stored at −80 °C until analysis. To lyse the cells, the samples were boiled for 2 min in 300 µL boiling buffer  and sonicated on ice. Samples were diluted 300-fold and 40 µL of the diluted samples were used to determine the ATP concentration using the ATP bioluminescence assay kit HS II  according to the manufacturer’s protocol with an Optocomp I luminometer . The ATP level is reported as µmol ATP/mg protein.The basal ATP levels of the stable cell lines expressing WT, K39R, K39Q, K39E Cytc, and EV were measured. Cells were seeded at 25,000 cells/well onto a 0.1% gelatin coated Seahorse XFe24 cell culture microplate  and cultured overnight in 250 µL/well growth media. The growth media was replaced with 675 µL/well seahorse media generated by dissolving 4.15 g DMEM powder  in 500 mL ddH2O, pH 7.4, sterile filtered , and supplemented with 10 mM glucose and 10 mM sodium pyruvate without phenol red, FBS, or sodium bicarbonate. After the media change, cells were incubated in a CO2 free incubator for 1 h, and then OCR and ECAR were measured in an XFe24 Seahorse extracellular flux analyzer according to manufacturer’s protocol. Mitochondrial stress test was performed via sequential injection of 1 µM oligomycin, 2.5 µM carbonylcyanide-3-chlorophenylhydrazone (CCCP), and 1 µM rotenone/antimycin A. Basal respiration, non-mitochondrial oxygen consumption, proton leak, ATP-coupled respiration, maximal respiration, and spare respiratory capacity were calculated according to manufacturer’s protocol. Briefly, non-mitochondrial oxygen consumption was calculated using the OCR values after the final injection of 1 µM rotenone and antimycin A, which inhibit complexes I and III, respectively. The basal respiration was calculated using the initial OCR values prior to any chemical injections minus the non-mitochondrial oxygen consumption. The proton leak was calculated using the OCR values after the injection of 1 µM oligomycin, which inhibits ATP synthase, minus the non-mitochondrial oxygen consumption. ATP-coupled respiration was calculated using basal respiration minus the proton leak. Maximal respiration was calculated using the OCR values after the injection of 2.5 µM CCCP, which is a mitochondrial uncoupler, minus the non-mitochondrial oxygen consumption. The spare respiratory capacity was calculated using maximal respiration minus the basal respiration. OCR and ECAR were normalized to total protein content in the well after the experiment to control for variation in cell number between cell lines. For post-mitochondrial stress test normalization, cells in each well were lysed using 25 µL RIPA lysis buffer (see above) supplemented with protease inhibitor cocktail and protein concentration was determined by DC protein assay kit . OCR is reported as pmol O2/minute/µg protein and the ECAR is reported as mpH/minute/µg protein.The oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of the stable cell lines expressing WT, K39R, K39Q, K39E Cytm) of the stable cell lines expressing WT, K39R, K39Q, K39E, and EV were measured using the JC-10 probe . Cells were seeded at 25,000 cells/well onto 0.1% gelatin coated Costar 96-well plates and cultured overnight in 100 µL/well growth media. The next day, cells were washed with 1x PBS and incubated for 30 min at 37 °C with 3 µM JC-10 in DMEM  without phenol red or FBS. After incubation, cells were washed twice with 1x PBS. JC-10 can selectively enter mitochondria where it exists as a monomer at low ΔΨm with primarily green fluorescence (excitation/emission of 485 nm/527 nm). When ΔΨm is high, JC-10 aggregates with primarily red fluorescence (excitation/emission of 485 nm/590 nm). Fluorescence was measured in PBS using a Synergy H1 microplate reader . ΔΨm is reported as the ratio of red/green fluorescence.The relative mitochondrial membrane potentials . MitoSOX can selectively enter mitochondria where it fluoresces red (excitation/emission of 510 nm/580 nm) after reaction with superoxide. Cells were seeded at 25,000 cells/well onto 0.1% gelatin coated Costar 96-well plates and cultured overnight in growth media. The next day, cells were washed with 1x PBS and incubated for 30 min at 37 °C with 5 µM mitoSOX in DMEM  without phenol red or FBS. After incubation, cells were washed twice with 1x PBS and fluorescence was measured in PBS using a Synergy H1 microplate reader. Mitochondrial ROS production is reported as a percentage of change in fluorescence/µg protein (arbitrary units/µg protein) compared to WT.The production of mitochondrial ROS of the stable cell lines expressing WT, K39R, K39Q, K39E Cytc, and EV were exposed to transient oxygen-glucose deprivation followed by reoxygenation (OGD/R) as previously described20. Cells were seeded and cultured according to the above protocols for JC-10 or MitoSOX. After culturing overnight, the growth media was exchanged for ischemia-mimetic DMEM media  without glucose, phenol red, or FBS that had been bubbled with 95% N2 and 5% CO2. Cells were incubated in ischemia-mimetic media at 37 °C for 90 min with 1% O2 and 5% CO2 in a hypoxia chamber controlled by ProOx 110 Compact O2 Controller  and ProCO2 120 Compact CO2 Controller . After ischemia, cells were reoxygenated via incubation in glucose-containing DMEM  without phenol red or FBS, containing the respective probe, at 37 °C for 30 min with 5% CO2. The JC-10 or MitoSOX experiments were then carried out as described in the methods above.To simulate ischemia/reperfusion injury in a cell culture model, stable cell lines expressing WT, K39R, K39Q, K39E Cytc, and EV were quantified after exposure to H2O2 or OGD/R using annexin V and propidium iodide (PI) staining. Annexin V is impermeable to the intact plasma membrane and binds phosphatidylserine, a lipid normally present on the inner leaflet of the plasma membrane which flips to the outer leaflet early during apoptosis. Therefore, annexin V is commonly used as a marker of early apoptotic cells. PI is also impermeable to the intact plasma membrane and is a DNA intercalator. During necrosis, holes in the plasma membrane allow PI to enter. Therefore, PI is commonly used as a marker of necrotic cells93. Cells that stain double positive for both annexin V and PI are late apoptotic cells. Cells (1 x 106) were seeded on 10 cm cell culture dishes and cultured overnight in growth media. The next day, cells were exposed to either H2O2 (400 µM for 16 h), OGD/R (16 h oxygen-glucose deprivation/1 h reoxygenation) as described above, or thapsigargin  (1 mM for 24 h). After 48 h from the initial seeding, cells were trypsinized, washed twice with 1x PBS, counted, and a total of 1 x 106 cells were resuspended in 1 mL 1x annexin V binding buffer from the FITC annexin V apoptosis detection kit I . A total of 450 µL cell suspension was incubated for 15 min with 6 µL annexin V and 6 µL PI. Data were collected using a CyFlow Space flow cytometer . The results were analyzed using FCS Express 7 software .Cell death levels of the stable cell lines expressing WT, K39R, K39Q, K39E Cyt6) were seeded onto 15 cm cell culture dishes  and cultured overnight. The next day, cells were treated with 1 μM staurosporine  for 8 h, which is known to be a potent inducer of intrinsic apoptosis47. Cells were washed with 1x PBS, harvested via trypsinization, and pelleted via centrifugation. The cell pellets were resuspended and lysed in 200 µL RIPA lysis buffer supplemented with protease inhibitor cocktail, sonicated, and centrifuged at 16,900 x g for 20 min at 4 °C to remove cell debris. Ninety μg cell lysates were run on a 10% tris-tricine SDS-PAGE gel, transferred, and blocked as described above. Membranes were incubated with either a 1:500 dilution of mouse anti-caspase-9 antibody  or a 1:1000 dilution of rabbit anti-caspase-3 antibody  in blocking reagent overnight at 4 °C. The next day, the membrane was incubated with a 1:8000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody or a 1:8000 dilution of donkey anti-rabbit IgG conjugated to horseradish peroxidase secondary antibody, respectively, for 1 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate. The next night, the membranes were incubated with a 1:5000 dilution of mouse anti-GAPDH antibody, as a loading control, in blocking reagent overnight at 4 °C. The next day, the membrane was incubated with a 1:8000 dilution of sheep anti-mouse IgG conjugated to horseradish peroxidase secondary antibody in blocking reagent for 1 h at room temperature. The blots were visualized using Pierce ECL western blotting substrate.Caspase cleavage of the cell lines stably expressing WT, K39R, K39Q, K39E, and EV were assessed via western blot. Cells . A student’s two-tailed t test assuming equal variance was used for COX activity (to compare porcine TA muscle samples), caspase-3 activity (to compare porcine TA muscle samples), and OGD/R (to compare normoxia and hypoxia results within each cell line). P values are indicated in the figure legends. The experiments were not randomized, and investigators were not blinded to allocation during experiments or outcome assessment.Data shown represent the mean, and error bars represent the standard deviation between independent trials for protein experiments or individual wells/cell culture dishes for cellular experiments. Statistical analyses of the data were performed using Graphpad Prism v9.4.1 . Most data were analyzed using one-way ANOVA comparing the mean of each column with the mean of the control column (WT) with the Dunnett post-hoc test unless otherwise specified. COX activity of the recombinant purified proteins was performed using one-way ANOVA as described above on the 25 μM CytFurther information on research design is available in the Supplementary InformationPeer review fileReporting summary


Various testing methods were applied to investigate the effect of various copper contents on the catalysts. The catalytic performance was evaluated by a fixed bed reactor. XRD, HRTEM and Raman spectra collectively indicated that a ZnO-ZrO2 solid solution catalyst with 3% Cu had a higher Cu dispersion, while the H2-TPR results confirmed that a catalyst with 3% Cu had more Cu active sites under low temperature H2 pretreatment. When the copper content increased to 5% and 10%, the catalyst showed a better Cu crystallinity and a worse Cu dispersion, which could have a negative effect. Therefore, the CO2 conversion and methanol yield with a 3% CuZnO-ZrO2 catalyst at 5 MPa, 250°C and 12 000 ml/(g h) increased by 8.6% and 7.6%, respectively. Moreover, the CH3OH selectivity and catalytic stability of the solid solution catalyst were better than those of the traditional CZA catalyst.Copper-doped ZnO-ZrO CO2 imissions . CO2 hydsolution . Methanosolution , olefinssolution  and DME solution , and cansolution .2O3 (CZA), Pd/Zn/CeO2 and Cu/Ni/CeO2 catalyst showed better performance. Pd/Zn/CeO2 is a noble metal catalyst and its large-scale application is limited by the higher cost, while the CZA catalysts with ultra-low cost have been extensively applied to CH3OH synthesis from hydrogenation [2 catalysts are also applied to methanol synthesis. Compared with alumina, zirconia has many excellent properties to provide copper dispersion sites and surface alkalinity, so the CuO/ZnO/ZrO2 catalysts could be more effective on the CO2 conversion to methanol [Among the reported catalysts, the Cu/ZnO/Algenation . Besidesmethanol . In ordemethanol , immersimethanol  and sol-methanol  have beeet al. developed a ZnO-ZrO2 solid solution with a CH3OH selectivity of 91% and CO2 conversion of 10%. The catalyst also showed excellent stability and provided new ideas. As we all know, the solid solution catalysts that have been reported at present are CeO2-based  and ZrO2-CeO2 solid solution catalysts have excellent CO catalytic oxidation activity [xZr1-x O2-based catalysts, noble metals usually act as active components [xZr1-xO2 showed a higher catalytic ability of CO oxidation and NO removal. For the copper oxide modified CexZr1-xO2 and CuO-CeO2 solid solution, the copper oxide species generally consist of three types and it has been widely accepted that highly dispersed CuO is more active than lattice doped and bulk phase CuO [2 conversion into methanol, while RWGS is the most likely secondary reaction. On the other hand, CO2 has strong chemical stability, so it is very important to study catalysts with fabulous catalytic activity and stability [2 solid solution, Li [et al. also reported a class of metal oxide solid solution catalysts: MaZrOx  for CO2 hydrogenation to methanol. Other solid solution catalysts, such as ZnO-ZrO2-MOx, were subsequently reported for the same application [Recently, Li  et al. dactivity . For Cexmponents , and meamponents . Among thase CuO ,19. Thertability . Apart ftion, Li  et al. alication .2 formed a solid solution structure. On this basis, several x% CuOZnO-ZrO2 were prepared by the co-precipitation (x% represents the molar percentage of copper). Generally, Cu2+ ions could embed into the lattice of CeO2 and form a solid solution at samll Cu/(Ce + Cu) proportions (less than 0.1). The ranges of copper doping content were less than 10% .In this paper, when the Zn content was 20% , ZnO and ZrO2 hydrogenation to methanol, and there are many studies on introducing other supports or promoters into Cu-Zn catalysts. In our work, CuZnO-ZrO2 solid solution catalyst is the focus of research, and CZA is prepared as the comparison catalyst. Although there are a lot of catalysts that appear to be the same or similar in composition in literatures, the solid solution structure and catalytic performance of the CuZnO-ZrO2 catalyst in our work is significantly different from the other catalysts. After the ZnOZrO2 solid solution catalyst is doped with copper, a ternary solid solution is formed, which can improve its CO2 conversion while maintaining relatively high methanol selectivity. This is also the novelty of this study.Cu-Zn binary catalysts have been widely used for CO2 hydrogenation to CH3OH of the catalysts were studied and discussed.The representative physical and chemical properties of the catalysts were tested by various techniques. The facilitation of Cu components and the effects of Cu content on CO. 2. 2.12 were synthesized by a coprecipitation method, then the copper-doped ZnO-ZrO2 catalysts (the Cu/Zn/Zr molar ratio is 0–0.1/1/4) were also prepared by co-precipitation. The specific synthesis procedures are as follows: 1.49 g Zn(NO3)2·6H2O, 8.59 g Zr(NO3)4·5H2O and 0.18 g Cu(NO3)2·3H2O  were dissolved in distilled water, then 0.5 mol l−1 of Na2CO3 was dispersed in the aforementioned solution under stirring at 70°C to form a precipitate. The pH during precipitation was kept at 7.5 under continuous stirring for 1 h at 70°C, and then it was incubated for 3 h at 70°C. When cooled, the precipitate was centrifuged, washed with deionized water and dried at 105°C overnight. Finally, the product was calcined at 500°C in the air for 3 h. Other catalysts with different copper contents were prepared following the same method. The CZA catalysts (with molar ratios of Cu/Zn/Al = 6/3/1) were also synthesized by co-precipitation and their preparation process is consistent with the above statement. The amount of reagent is 7.2 g Cu(NO3)2·3H2O, 4.5 g Zn(NO3)2·6H2O, and 1.9 g Al(NO3)3·9H2O. For comparison, all reaction conditions, such as reaction temperature, reaction time and pH value, are completely consistent with the above experiments.Bimetallic oxide catalysts with various molar ratios of Zn-Zr, pure ZnO and ZrO. 2.2α radiation (λ = 0.15416 nm), operating at 40 kv and 40 mA. N2 adsorption-desorption isotherms were obtained at −196°C by a Beckman Coulter SA 3100 type specific surface area and aperture measurement instrument  and the surface areas of the catalysts were calculated by the BET method. TG curves of the catalyst precursors was collected by an STA 449 TG-DSC synchronous thermal analyzer . Raman spectra were recorded by a Raman microscope with a laser beam (λ = 325 nm). XPS spectra were used to measure the element composition and valence distribution of the catalysts (C1s peak at 284.8 eV).The crystalline phase of the catalysts was investigated by a Panalytical X'Pert Pro X-ray diffractometer with Cu K2-TPR was used to detect the reducibility. First, the catalyst in the tube was heated to 130°C in argon (50 ml min−1) keeping the samples at pretreatment for 2 h. Next, they were cooled to 50°C, then the samples were heated to 800°C in a 10% H2/Ar mixture and hydrogen consumption was measured by the AutoChemII2920 with a TCD detector. TEM images were observed on a TEM instrument at 200 kV accelerating voltage.H. 2.32 hydrogenation to CH3OH was tested in a tubular reactor. The catalyst  was pretreated for 4 h in a 50% H2/N2 stream (20 ml min−1 and 0.2 MPa). The temperature was raised to 280°C, maintained for 4 h, and then cooled down. Next, the reaction was conducted at 1–5 MPa and 12 000 ml/(g h) GHSV, the reaction temperature was 190°C to 310°C and the volume ratio of H2 : CO2 : N2 was 72 : 24 : 4. The reaction products were collected after 3 h and then the reaction gas was analysed. Conversion of CO2, CH3OH selectivity and yield were calculated by the following equations (2.1)–(2.4).COAi means the peak area of the corresponding product and fi means its correction factor.. 3. 3.1. 3.1.1a exhibits the XRD of the ZnO-ZrO2 with various Zn contents. When the Zn molar content was 20%, the ZnO-ZrO2 formed a solid solution. Figure 1b shows the XRD of Cu-doped catalysts. Four sharp diffraction peaks are assigned to the crystal planes of t-ZrO2, respectively (JCPDS Card No. 50-1089) [θ of 36.1°(JCPDS Card No. 03-0879) in the 5% CuZnO-ZrO2 catalyst and in the 10% CuZnO-ZrO2 catalyst, which reveals that the Cu species existed in large particles of CuO [Figure 1c shows the XRD of the t-ZrO2 (011) peak in all of the catalysts. It was found that the characteristic peak of ZrO2 (011) became broader, while the peak position offset to a smaller angle. This migration phenomenon may be due to the lattice distortion caused by copper ions entering the ZnO-ZrO2 tetragonal lattice. In 2, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 catalysts that were calculated by the Scherrer equation were 11.3 nm, 11.8 nm and 8.6 nm, respectively. The Raman spectra of the 0% CuZnO-ZrO2, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 are shown in figure 1d. The peak of 0% CuZnO-ZrO2 at 564 cm−1 proves that there is a solid solution structure. Similarly, the peak at approximately 564 cm−1 was observed and CuO and Cu2O weren't observed, which proved that the Cu-Zn-Zr-O ternary solid solution was generated in the 3% CuZnO-ZrO2 catalyst. However, the peaks at 295, 331 and 625 cm−1 are assigned to the Raman-active Ag, 2Bg symmetry of CuO that existed in the 10% CuZnO-ZrO2 catalyst. The scanning results of the Raman spectra fit those of XRD.Figure 150-1089) . The difs of CuO . Figure . 3.1.22, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 are shown in figure 2a. The TG curve of the catalyst precursor consists of four stages: before 100°C, the weight loss was recognized as the loss of physically absorbed water, corresponding to the endothermic peaks in figure 2b–d. The decomposition of zinc carbonate 100–250°C corresponds to the endothermic peaks in figure 2b–d. Copper carbonate precipitates decompose at 250–450°C without an obvious endothermic peak in figure 2c, while significant endothermic peaks appear in figure 2d, which implies that there is copper in precursor of 10% CuZnO-ZrO2. A significant weight loss between 500 and 550°C reveals the formation of a solid solution structure in the precursors of the 0% CuZnO-ZrO2 and 3% CuZnO-ZrO2 catalysts, corresponding to the exothermic peak in figure 2b,c, while there was no obvious exothermic peak in the 10% CuZnO-ZrO2 precursor in figure 2d [2, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 precursors were 19.6%, 21.4% and 23.4%, respectively  peak, while the average size of the 10% CuZnO-ZrO2 catalyst was bigger than the crystalline size calculated by the Scherrer equation.TEM morphology and size distribution of the catalysts are depicted in figure 42 catalyst are distributed uniformly. The HRTEM images of the 0% CuZnO-ZrO2, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 catalysts are collected in figure 4d–f. The distance between two adjacent lattice stripes was approximately 0.294 nm, which is equal to the spacing of the (011) plane of t-ZrO2 [2O and ZnO were not found, which corresponds to the XRD results: no diffraction peaks of the ZnO and CuO or Cu2O can be observed in the patterns.It can be concluded that the nanoparticles of the 3% CuZnO-ZrO. 3.1.5a displays XPS spectra of the 0% CuZnO-ZrO2, 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 catalysts, which confirm the coexistence of Zr, C, O, Cu, Zn and Na species. The copper content on the surface was higher than the experimental addition amount in 3% CuZnO-ZrO2, while the copper content of the surface was less than the experimental addition amount in 10% CuZnO-ZrO2. The Zn content was less than the theoretical value in the 0% CuZnO-ZrO2 and 3% CuZnO-ZrO2 catalysts, indicating that the ZnO surface was partly covered in CuO and ZrO2. Conversely, enriched Zn on the surface was observed in the 10% CuZnO-ZrO2 catalysts, and analogous results were reported for other Cu-Zn catalysts [Figure 5atalysts .Figure 5/2 at 182 eV can be assigned to ZnO and ZrO2, respectively [3/2 XPS spectra are exhibited in figure 5b. The binding energy of 10% CuZnO-ZrO2 is greater than 3% CuZnO-ZrO2, and the peak at 932.1 eV corresponds to Cu0 or Cu+, which indicates a strong effect between Cu and the solid solution. The peak at 933.8 eV of 3% CuZnO-ZrO2 and the peak at 933.4 eV and 935.7 eV of 10% CuZnO-ZrO2 correspond to Cu2+ [The XPS data are listed in ectively . The Cu . 3.1.62-TPR profiles were shown in 2 and 10% CuZnO-ZrO2 catalysts than for the 0% CuZnO-ZrO2 catalyst, which indicates the solid solutions containing copper are more easily reduced than a copper free system. Both the 3% CuZnO-ZrO2 and 10% CuZnO-ZrO2 catalysts exhibited a broad reduction peak at 250–350°C as shown in the inset of 2, the first peak, the second peak and the third peak are designated as α, β and γ peak, respectively, where the α peak represents the CuO interacting with CeO2, the β peak originates from the deoxidation of highly dispersed CuO granules, and the γ peak is related to the deoxidation of bulk CuO [2 catalyst, the α and β peaks of the 3% CuZnO-ZrO2 catalyst both moved to a lower temperature and the γ peak only existed in 10% CuZnO-ZrO2, which corresponds to the results of XRD, Raman and other characterizations.Hbulk CuO . Compare. 3.2. 3.2.12 hydrogenation on the CuZnO-ZrO2 solid solution is shown in 2 conversion and CH3OH yield occurred when the copper addition amount was lower than 3%, while a decrease appeared when the copper amount was between 3% and 10%. However, the methanol selectivity did not change sharply with the copper content. A maximum catalytic activity was observed at a copper content of 3% in the solid solution catalyst. It could be concluded that the CO2 conversion increased significantly when copper was doped into the solid solution catalyst, and the optimal addition amount was 3%.The performance of CO. 3.2.2a–c, CO2 conversion increased with temperature, attended by a decline of CH3OH selectivity and an increase of CO selectivity, which is due to the competition between CH3OH synthesis and RWGS. The CH3OH synthesis is an exothermic reaction, but RWGS is the opposite. As seen in figure 8a, CO2 conversion of the traditional CZA catalyst was greater than the copper-doped ZnO-ZrO2 solid solution, while the CH3OH selectivity was the opposite (figure 8b). Compared with the solid solution catalysts, the CH3OH selectivity of the CZA catalyst decreased significantly with temperature. The solid solution catalyst in a high temperature reaction zone still maintains high methanol selectivity, which provides a theoretical basis C–C coupling reactions on the modified catalyst [figure 8d, the CH3OH yield has a maximum value for all of the catalysts. However, the maximum CH3OH yield for each of the catalysts corresponds to a different critical temperature, which is 250°C for 3% CuZnO-ZrO2, 270°C for 10% CuZnO-ZrO2, and 230°C for CZA catalyst. Clearly, the maximum CH3OH yield reveals the critical point of the transformation from dynamics to thermodynamics [The catalytic performance of the temperature dependence (210–310°C) is shown in figure 8catalyst . As showdynamics . When th. 3.2.32 hydrogenation on ZnO-ZrO2 solid solution catalysts with various copper contents and CZA catalysts was studied in the reaction pressure range of 1–5 Mpa, as shown in 2 conversion, CH3OH selectivity and yield increased with reaction pressure , attended by a decline of CO selectivity (c). The number of molecules is reduced in CO2 hydrogenation to CH3OH, so the catalytic reaction will move into a positive direction with an increasing reaction pressure, and catalytic activity will increase significantly in the meantime. At 5 MPa, the CO2 conversion is 17.7%, the methanol selectivity is 84.1%, and then the highest methanol yield of 14.9% was obtained using 3% CuZnO-ZrO2. Compared with the ZnO-ZrO2 solid solution without copper addition, the CO2 conversion and CH3OH yield were increased by 8.6% and 7.6%, respectively. The highest CO2 conversion of 19.7% was found with the CZA catalyst, while the methanol selectivity was relatively low, and thus the methanol yield was not high.COpressure a,b,d, atectivity c. The nu. 3.2.43OH synthesis. The effect of the reaction time on the catalytic stability was investigated for 100 h. In 2 conversion of CZA catalyst decreased from 16.27% to 8.92% with an attenuation rate of 45.2%, while the methanol selectivity decreased from 36.1% to 29.2% with an attenuation rate of 19.1%. The marked deactivation phenomenon of the CZA catalyst can be attributed to sintering of Cu particles. Howcver, the 3% CuZnO-ZrO2 catalyst had the highest stability for CO2 methanation to CH3OH and could run for 100 h almost without any inactivation.The stability of the catalyst is also a key performance in CH. 3.2.52 catalyst in our work is significantly different from the other catalysts. Here, the catalysts closely related to this paper, such as CuZnZr, CuZnAl catalysts prepared by different methods and other solid solution catalysts, were listed in Although there are a lot of catalysts that appear to be the same or similar in composition in literature, the solid solution structure and catalytic performance of the CuZnO-ZrO2 conversion and CH3OH selectivity of the reported solid solution catalysts ZnZr, Cd/GaZr and MgZnZr are 10–12.9% and 80–86% respectively at 3–5 MPa, 320°C and H2: CO2 molar ratio of =3 : 1 [2 conversion of CuZnZr catalysts prepared by different methods under different conditions varies from 12.0% to 18.7%, and the CH3OH selectivity varies from 45% to 71.1% [2 conversion of CuZnAl catalysts prepared by different methods under different conditions varies from 11% to 25%, and the CH3OH selectivity varies from 26% to 75% [3OH selectivity varies from 28% to 54% at the pressure range of 3–5 MPa, temperature range of 210–250°C, H2: CO2 molar ratio range of 5 : 3–3 : 1 and flow rate range of 500–1000 NmL gcat−1 min−1 [As shown in f =3 : 1 ,21,22. Tto 71.1% –40. The % to 75% –45. In a−1 min−1 .2 conversion and CH3OH selectivity of CZA catalyst are 16.27% and 36.1% respectively at 2 MPa, 250°C, 12 000 ml/(g h) and H2: CO2 molar ratio of = 3 : 1, which is roughly in line with the range reported in the above literature. The CO2 conversion and CH3OH selectivity of CuZnO-ZrO2 solid solution catalyst is 12.4–19.7% and 70.2–81.4% respectively at 2–5 MPa, 250°C, 12 000 ml/(g h) and H2: CO2 molar ratio of = 3 : 1, whose catalytic activity level appears to be above average. Due to the different reaction conditions used in various studies, the comparison of catalytic performance has some reference value but cannot be completely trusted.In our study, the CO. 42 solid solution catalysts with various copper contents were synthesized for CO2 hydrogenation to CH3OH. The XRD, HRTEM and Raman spectra analysis results showed that there is a solid solution structure in the 3% CuZnO-ZrO2 catalyst, and its specific surface area is 38.408 m2 g−1, higher than those of the 0% CuZnO-ZrO2 and 10% CuZnO-ZrO2 catalysts. The H2-TPR analysis proved that the 3% CuZnO-ZrO2 catalyst exhibited higher catalytic performance at a relatively lower reaction temperature. As against 10% CuZnO-ZrO2, 3% CuZnO-ZrO2 contains more dispersed copper species, whose catalytic activity is higher than that of the bulk CuO species. The XPS analysis indicates a strong interaction between Cu and the ZnO-ZrO2 solid solution. The CO2 conversion and CH3OH yield of the 3% CuZnO-ZrO2 reached 17.7% and 14.9%, respectively ), while those of the 0% CuZnO-ZrO2 were 9.1% and 7.3%, respectively. Compared with the traditional CZA catalyst, the Cu-doped solid solution had higher methanol selectivity and better high temperature resistance.ZnO-ZrO