1. The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor.
- Author
-
Rao G, Pattenaude SA, Alwan K, Blackburn NJ, Britt RD, and Rauchfuss TB
- Subjects
- Bacterial Proteins metabolism, Catalysis, Catalytic Domain, Cysteine metabolism, Electron Spin Resonance Spectroscopy, Iron metabolism, Organometallic Compounds metabolism, Sulfur metabolism, Bacterial Proteins chemistry, Cysteine chemistry, Hydrogenase chemistry, Iron chemistry, Organometallic Compounds chemistry, Sulfur chemistry
- Abstract
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 with13 CN-containing carrier affords13 CN-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 a13 C3 ,15 N-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., Competing Interests: The authors declare no competing interest.- Published
- 2019
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