Marius Horch

Probing the active site of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 by in situ EPR and FTIR spectroscopy.

[NiFe]-hydrogenases catalyze the reversible cleavage of dihydrogen into two protons and two electrons. This process plays an important role in the energy metabolism of many microorganisms. For most [NiFe]-hydrogenases, the process of H2 cycling is extremely sensitive to molecular oxygen as O2 exhibits a high affinity to the active site. However, some organisms are capable of catalyzing H2 cycling even at ambient oxygen levels. Notably, the b-proteobacterium Ralstonia eutropha H16 (Re) harbors three different [NiFe]hydrogenases, all of which display a remarkable oxygentolerance. The underlying molecular mechanisms are not yet fully understood. For the regulatory hydrogenase (RH) of Re, a narrow gas tunnel is thought to restrict O2 access to the active site. The Re membrane-bound hydrogenase (MBH) has a high redox potential FeS cluster in close proximity to the active site, a property that might be related to the observation that O2-inhibited MBH re-activates rapidly at high potentials. The soluble hydrogenase (SH) of Re is a cytoplasmic NAD-reducing six-subunit enzyme that is closely related to cyanobacterial bidirectional [NiFe]-hydrogenases. 7] For purified SH, a modified catalytic site was proposed on the basis of numerous biochemical and spectroscopic studies. 8, 9] In contrast to “standard” [NiFe]-hydrogenases, in which the active site iron is kept in the low-spin iron(II) state by one carbonyl and two cyanide ligands, Fourier transform infrared (FTIR) spectroscopy and concomitant chemical analysis suggested one additional cyanide bound to each metal ion of the catalytic center. The nickelbound cyanide ligand has been proposed to prevent the formation of the so-called Niu-A state, which is the most oxidized, O2-inactivated state in [NiFe]-hydrogenases. [9]

NAD(H)-coupled hydrogen cycling – structure–function relationships of bidirectional [NiFe] hydrogenases

Hydrogenases catalyze the activation or production of molecular hydrogen. Due to their potential importance for future biotechnological applications, these enzymes have been in the focus of intense research for the past decades. Bidirectional [NiFe] hydrogenases are of particular interest as they couple the reversible cleavage of hydrogen to the redox conversion of NAD(H). In this account, we review the current state of knowledge about mechanistic aspects and structural determinants of these complex multi‐cofactor enzymes. Special emphasis is laid on the oxygen‐tolerant NAD(H)‐linked bidirectional [NiFe] hydrogenase from Ralstonia eutropha.

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.

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 in vitro and in vivo 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.

Revealing the Absolute Configuration of the CO and CN− Ligands at the Active Site of a [NiFe] Hydrogenase

Combined molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the crystal structure of the reduced membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha to determine the absolute configuration of the CO and the two CN(-) ligands bound to the active-site iron of the enzyme. For three models that include the CO ligand at different positions, often indistinguishable on the basis of the crystallographic data, we optimized the structures and calculated the ligand stretching frequencies. Comparison with the experimental IR data reveals that the CO ligand is in trans position to the substrate-binding site of the bimetallic [NiFe] cluster.

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 sensitivity 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.

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

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.

Combining Spectroscopy and Theory to Evaluate Structural Models of Metalloenzymes: A Case Study on the Soluble [NiFe] Hydrogenase from Ralstonia eutropha

Hydrogenases catalyse the reversible cleavage of molecular hydrogen into protons and electrons. While most of these enzymes are inhibited under aerobic conditions, some hydrogenases are catalytically active even at ambient oxygen levels. In particular, the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 couples reversible hydrogen cycling to the redox conversion of NAD(H). Its insensitivity towards oxygen has been formerly ascribed to the putative presence of additional cyanide ligands at the active site, which has been, however, discussed controversially. Based on quantum chemical calculations of model compounds, we demonstrate that spectroscopic consequences of the proposed non-standard set of inorganic ligands are in contradiction to the underlying experimental findings. In this way, the previous model for structure and function of this soluble hydrogenase is disproved on a fundamental level, thereby highlighting the efficiency of computational methods for the evaluation of experimentally derived mechanistic proposals.

An S‐Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O2‐Tolerant Hydrogenase

To understand the molecular details of O2 -tolerant hydrogen cycling by a soluble NAD+ -reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S-oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen-inhibited [NiFe] active site. This compound and its non-S-oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S-oxygenated intermediates in hydrogenases and similar enzymes.

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures.