Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD⁺‑Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H₂ oxidation and production. The soluble NAD⁺-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H₂ with the reduction of NAD⁺ and displays a unique O₂ tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained <i>in vitro</i> and <i>in vivo</i> could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O₂ catalytically by means of an NADH-dependent (per)­oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H₂-conversion even in the presence of O₂

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

[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

Impact of the Iron–Sulfur Cluster Proximal to the Active Site on the Catalytic Function of an O₂‑Tolerant NAD⁺‑Reducing [NiFe]-Hydrogenase

The soluble NAD⁺-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O₂-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O₂ tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron–sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O₂-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O₂ sensitity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O₂-tolerant SH representatives did not greatly affect the H₂-oxidizing activity in the presence of O₂. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron–sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H₂/D⁺ exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from <i>Ralstonia eutropha</i> with O₂ and CO

The regulatory hydrogenase (RH) from <i>Ralstonia eutropha</i> acts as the H₂-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H₂. The H₂ oxidation activity, which was so far determined <i>in vitro</i> with artificial electron acceptors, has been considered to be insensitive to O₂ and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site “gate” gas access, preventing molecules larger than H₂ interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O₂ is in fact an inhibitor of H₂ oxidation by the RH, and that both H⁺ reduction and H₂ oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O₂ arises due to interaction of O₂ with the active site. Using protein film infrared electrochemistry (PFIRE) under H₂ oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases

Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase

<div><p>Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H₂ conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.</p></div