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[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2into 2e−and 2H+under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN−molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN–) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN)2(CO) cofactor requires the activity of at least six maturation proteins, designated HypA–F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivodata indicated that HypX releases CO using N10-formyltetrahydrofolate (N10-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N10-formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitroevidence that purified HypX first transfers the formyl group of N10-formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situto investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator(MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

The catalytic properties of hydrogenases are nature’s answer to the seemingly simple reaction H2⇌ 2H++ 2e–. Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H2-activating [NiFe] site and the small subunit contains iron–sulfur clusters mediating e–transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O2. Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O2-tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H2as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H2activation was performed by preHoxG even in the presence of O2, although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O2was absent. These findings challenge the current understanding of O2tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.

Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the “Knallgas” bacterium Ralstonia eutrophaby relating its electrochemical activity for hydrogen oxidation to the interface properties.