The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO)2(CN)] organometallic precursor.
The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe-Fe binuclear subcluster: CN-, CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren't fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO)2(CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO)2(CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13CN-containing carrier affords 13CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a 13C3,15N-Cys-labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1-Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.
['Rao|Guodong|G|0000-0001-8043-3436', 'Pattenaude|Scott A|SA|', 'Alwan|Katherine|K|', 'Blackburn|Ninian J|NJ|0000-0002-9755-7838', 'Britt|R David|RD|0000-0003-0889-8436', 'Rauchfuss|Thomas B|TB|0000-0003-2547-5128']
D001426:Bacterial Proteins D002384:Catalysis D020134:Catalytic Domain D003545:Cysteine D004578:Electron Spin Resonance Spectroscopy D006864:Hydrogenase D007501:Iron D009942:Organometallic Compounds D013455:Sulfur
cysteine H-cluster biosynthesis iron carbonyl cyanides