Expanding the scope of resonance Raman spectroscopy in hydrogenase research: New observable states and reporter vibrations.

Oxygen-tolerant [NiFe] hydrogenases are valuable blueprints for the activation and evolution of molecular hydrogen under application-relevant conditions. Vibrational spectroscopic techniques play a key role in the investigation of these metalloenzymes. For instance, resonance Raman spectroscopy has been introduced as a site-selective approach for probing metal-ligand coordinates of the [NiFe] active site and FeS clusters.

Light-Induced Electron Transfer in a [NiFe] Hydrogenase Opens a Photochemical Shortcut for Catalytic Dihydrogen Cleavage.

[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen-tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Ni-C and Ni-SR'', were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron-sulfur clusters of the enzyme.

Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)] in Polar and Protic Solvents.

The [CpFe(CO)(CN)] unit is an excellent structural model for the Fe(CO)(CN) moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)] () in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of in HO, DO, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN) unit.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD-reducing [NiFe] hydrogenases are valuable biocatalysts for H-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O and elevated temperatures, the soluble NAD-reducing [NiFe] hydrogenase from (SH) is O-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications.

Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.

To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O -tolerant [NiFe]-hydrogenases were investigated as model systems.

Shedding Light on Proton and Electron Dynamics in [FeFe] Hydrogenases.

[FeFe] hydrogenases are highly efficient catalysts for reversible dihydrogen evolution. H turnover involves different catalytic intermediates including a recently characterized hydride state of the active site (H-cluster). Applying cryogenic infrared and electron paramagnetic resonance spectroscopy to an [FeFe] model hydrogenase from (HydA1), we have discovered two new hydride intermediates and spectroscopic evidence for a bridging CO ligand in two reduced H-cluster states.

Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.

Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail.

X-ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases.

[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H ). However, structural determinants of efficient H binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H -binding [NiFe] intermediate.

Rational redox tuning of transition metal sites: learning from superoxide reductase.

Using superoxide reductase as a model system, a computational approach reveals how histidine tautomerism tunes the redox properties of metalloenzymes to enable their catalytic function. Inspired by these experimentally inaccessible insights, non-canonical histidine congeners are introduced as new versatile tools for the rational engineering of biological transition metal sites.

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H-driven NAD-reduction in the presence of O.

Biocatalysts that mediate the H-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 O, 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-1 (Ht).

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD and at various pH values.

The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K  ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations.

An S-Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O -Tolerant Hydrogenase.

To understand the molecular details of O -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.

Investigation of the NADH/NAD ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H-driven production of biodegradable polymers and hydrocarbons.

Domain motions and electron transfer dynamics in 2Fe-superoxide reductase.

Superoxide reductases are non-heme iron enzymes that represent valuable model systems for the reductive detoxification of reactive oxygen species. In the present study, we applied different theoretical methods to study the structural dynamics of a prototypical 2Fe-superoxide reductase and its influence on electron transfer towards the active site. Using normal mode and essential dynamics analyses, we could show that enzymes of this type are capable of well-defined, electrostatically triggered domain movements, which may allow conformational proofreading for cellular redox partners involved in intermolecular electron transfer.

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.

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

The regulatory hydrogenase (RH) from Ralstonia eutropha 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 in vitro 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.

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O-tolerant NAD-reducing [NiFe] hydrogenase.

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD-reducing soluble hydrogenase (SH) from is capable of H conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme.

Microporous polymer network films covalently bound to gold electrodes.

Covalent attachment of a microporous polymer network (MPN) on a gold surface is presented. A functional bromophenyl-based self-assembled monolayer (SAM) formed on the gold surface acts as co-monomer in the polymerisation of the MPN yielding homogeneous and robust coatings. Covalent binding of the films to the electrode is confirmed by SEIRAS measurements.

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.

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.

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.

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).

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.