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

Abstract

[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 (Fed). 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 H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

“…Strong bands between 500–600 cm -1 are modes mostly of Fe-CO bending and stretching character; although their profiles are similar under H vs D conditions, there are indeed subtle changes that derive from coupling with Fe-H/D motion. 21 …”

“…Four schematic alternatives on H hyd based on earlier proposals 21 are shown in Figure 5F. Two of them, designated H hyd-A and H hyd-E , assume an uncharged bridgehead amino group, in either ‘axial’ −N ADT H A− (H A pointing toward Fe d ) or ‘equatorial’ −N ADT H E− (H E pointing away from Fe d ) conformation.…”

“…The corresponding red-shifted Fe d -D h modes were associated with the perturbed Fe d/p –CO bands at 625 and 564 cm -1 under D 2 /D 2 O conditions. 21 The Fe d -H h vibrations at 670 and 727 cm -1 were assigned, respectively, to H − motion perpendicular to and within the approximate mirror symmetry plane of [2Fe] H , which passes through Fe p , Fe d , and H h (Fe p Fe d H h plane). Here we focus on the NRVS spectra for the H hyd species from Cr HydA1 and Dd HydAB artificially maturated with [2 57 Fe] H .…”

NRVS for Fe in Biology: Experiment and Basic Interpretation

“…37 Further as the systems of interest have become more complicated, with multiple FeS clusters of differing composition, innovative strategies of site-specific 57 Fe labeling have been implemented that target maturation 28 or a modified cofactor insertion. 38 Since its inception two decades ago, improvements in synchrotron flux, resolution, and sample preparation have allowed for the NRVS investigations of weaker features and more dilute biological samples. The technique is continuing to mature and is solving questions in ways that resonance Raman and infrared spectroscopies cannot.…”

“…As such, the extreme specificity of 57 Fe NRVS for only 57 Fe motion, makes the perfect tool to study vibrational dynamics of the critical Fe-containing components of the biomolecule. Without contribution from the biological sample background, NRVS enjoys very little spectroscopic noise, even modes of Fe hydrides within enzymes 3–4 have been observed – which are typically difficult to directly identify with other techniques. 5–6 The selection rules – or comparatively lack thereof – allows for 57 Fe NRVS to reveal both IR- and Raman- active modes that contain 57 Fe motion – thus providing a complete picture for Fe vibration.…”