The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H‑Cluster

Three maturase enzymesHydE, HydF, and HydGsynthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe­(CO)₂(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe­(CO)₂(CN)] product. These findings support a mechanistic framework in which a [(Cys)­Fe­(CO)₂(CN)]⁻ species is a key intermediate in H-cluster maturation

Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [⁵⁷Fe₂(adt)(CN)₂(CO)₄]^2–

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively ⁵⁷Fe-labeled binuclear subsite is described. The precursor [⁵⁷Fe₂(adt)­(CN)₂(CO)₄]^2– was synthesized from the ⁵⁷Fe metal, S_8, CO, (NEt₄)­CN, NH₄Cl, and CH₂O. (Et₄N)₂[⁵⁷Fe₂(adt)­(CN)₂(CO)₄] was then used for the maturation of the [FeFe] hydrogenase HydA1 from <i>Chlamydomonas reinhardtii</i>, to yield the enzyme selectively labeled at the [2Fe]_H subcluster. Complementary ⁵⁷Fe enrichment of the [4Fe-4S]_H cluster was realized by reconstitution with ⁵⁷FeCl₃ and Na₂S. The H_ox-CO state of [2⁵⁷Fe]_H and [4⁵⁷Fe-4S]_H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy

Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H_hyd) in the [FeFe]-hydrogenases from both <i>Chlamydomonas reinhardtii</i> and <i>Desulfovibrio desulfuricans</i> using ⁵⁷Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe–H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe–H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that H_hyd is the catalytic state one step prior to H₂ formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H₂ bond formation by [FeFe]-hydrogenases

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]_H cluster linked through a bridging cysteine to a [2Fe]_H subsite coordinated by CN^– and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fe_d). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle