4 research outputs found
The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H‑Cluster
Three maturase enzymesHydE,
HydF, and HydGsynthesize
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)<sub>2</sub>(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)<sub>2</sub>(CN)] product. These findings support a mechanistic framework in
which a [(Cys)Fe(CO)<sub>2</sub>(CN)]<sup>−</sup> species is
a key intermediate in H-cluster maturation
Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [<sup>57</sup>Fe<sub>2</sub>(adt)(CN)<sub>2</sub>(CO)<sub>4</sub>]<sup>2–</sup>
The preparation and spectroscopic
characterization of a CO-inhibited
[FeFe] hydrogenase with a selectively <sup>57</sup>Fe-labeled binuclear
subsite is described. The precursor [<sup>57</sup>Fe<sub>2</sub>(adt)(CN)<sub>2</sub>(CO)<sub>4</sub>]<sup>2–</sup> was synthesized from
the <sup>57</sup>Fe metal, S<sub>8,</sub> CO, (NEt<sub>4</sub>)CN,
NH<sub>4</sub>Cl, and CH<sub>2</sub>O. (Et<sub>4</sub>N)<sub>2</sub>[<sup>57</sup>Fe<sub>2</sub>(adt)(CN)<sub>2</sub>(CO)<sub>4</sub>] 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]<sub>H</sub> subcluster. Complementary <sup>57</sup>Fe enrichment of the
[4Fe-4S]<sub>H</sub> cluster was realized by reconstitution with <sup>57</sup>FeCl<sub>3</sub> and Na<sub>2</sub>S. The H<sub>ox</sub>-CO
state of [2<sup>57</sup>Fe]<sub>H</sub> and [4<sup>57</sup>Fe-4S]<sub>H</sub> HydA1 was characterized by Mössbauer, HYSCORE, ENDOR,
and nuclear resonance vibrational spectroscopy
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]<sub>H</sub> cluster linked through a bridging cysteine to a [2Fe]<sub>H</sub> subsite coordinated by CN<sup>–</sup> 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<sub>d</sub>).
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
Reaction Coordinate Leading to H<sub>2</sub> 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<sub>hyd</sub>) in the
[FeFe]-hydrogenases from both <i>Chlamydomonas reinhardtii</i> and <i>Desulfovibrio desulfuricans</i> using <sup>57</sup>Fe 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<sub>hyd</sub> is the catalytic state one step prior to H<sub>2</sub> formation. The observed vibrational spectrum, therefore,
provides mechanistic insight into the reaction coordinate for H<sub>2</sub> bond formation by [FeFe]-hydrogenases