Resonance Raman Spectroscopy on [NiFe] Hydrogenase Provides Structural Insights into Catalytic Intermediates and Reactions

Abstract

[NiFe] hydrogenases catalyze the reversible cleavage of hydrogen and, thus, represent model systems for the investigation and exploitation of emission-free energy conversion processes. Valuable information on the underlying molecular mechanisms can be obtained by spectroscopic techniques that monitor individual catalytic intermediates. Here, we employed resonance Raman spectroscopy and extended it to the entire binuclear active site of an oxygen-tolerant [NiFe] hydrogenase by probing the metal-ligand modes of both the Fe and, for the first time, the Ni ion. Supported by theoretical methods, this approach allowed for monitoring H-transfer from the active site and revealed novel insights into the so far unknown structure and electronic configuration of the hydrogen-binding intermediate of the catalytic cycle, thereby providing key information about catalytic intermediates and reactions of biological hydrogen activation.

Resonance Raman spectroscopy of Fe–S proteins and their redox properties

“…Being sensitive to the type, ligands and configuration of a cluster, RR spectroscopy has been playing an important part in these discoveries. Some of the recent achievements include the evidence for the presence of a [2Fe–2S] 2+ cluster in a kinase/phosphatase Asp1 that regulates cell morphogenesis in yeasts [9], characterization of the [4Fe–4S] 2+ cluster in HydF, a protein involved in the maturation of organometallic H cluster of Fe–Fe hydrogenase [4] and elucidation of the missing pieces (i.e., the transient catalytic intermediates) of the catalytic cycle puzzle in hydrogenases [7, 82], which provide the key information about biological hydrogen activation. We believe that RR spectroscopy has a bright future in illuminating the structure and function of Fe–S clusters that are still to come to our lab benches.…”

“…RR has recently revealed structural insights into catalytic intermediate species in several of these enzymes [7, 82, 83]. Depending on the type and the source, hydrogenases can carry a variable number of different Fe–S clusters that are either essential for the ET and/or together with the binuclear center, constitute the active site.…”

“…In combination with theoretical approaches and FTIR spectroscopy, RR provided molecular details on the heterobimetallic active site and the Fe–S clusters in regulatory NiFe hydrogenase (RH) in oxidized and H 2 -reduced states. It has been used to probe the [4Fe–4S] 2+ clusters, which revealed bands below 400 cm −1 that diminish upon H 2 -incubation, indicating reduction to the RR-silent [4Fe–4S] 1+ state, and to simultaneously monitor the active site (i.e., Fe–CN and Fe–CO stretching modes) that show bands at higher (400–600 cm −1 ) frequencies [82]. Similarly, RR spectra of membrane-bound hydrogenase, from which the contributions of the Fe–S clusters were excluded by H 2 -reduction, revealed modes originating from the heterobimetallic active site that are sensitive to its structure [83].…”

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution

“…hydrogenases. 10–16 In the present study, we used this technique for the first time to probe the H-cluster of an [FeFe] hydrogenase under different redox conditions. Here, we chose HydA1 from the green alga Chlamydomonas reinhardtii as an ideal model system for spectroscopic studies, as it contains only the H-cluster and no additional cofactors that could obscure the spectra.…”

Nature's masterclass

“…10 The variability of the iron-sulfur clusters in MBH is a confounding factor, so the researchers used a simpler hydrogenase from the same organism instead, namely the regulatory hydrogenase (RH). This enzyme also splits hydrogen, but only in order to sense its concentration, and to upregulate the production of the main hydrogenase enzyme when it is needed.…”