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Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations.

ABSTRACT: The catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H₂/H⁺ cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit (HoxC) of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure resting redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, ⁵⁷Fe-labeled HoxC provided an unprecedented insight into the [NiFe] site modes, revealing their contributions in a spectral range otherwise superimposed by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data provide structural descriptions of the previously uncharacterized hydroxy- and water-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site.

SUBMITTER: Caserta G

PROVIDER: S-EPMC8179317 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chemically synthesized compounds that are capable of facilitating the reversible splitting of dihydrogen into protons and electrons are rare in chemists' portfolio. The corresponding biocatalysts - hydrogenases - are, however, abundant in the microbial world. [NiFe]-hydrogenases represent a major subclass and display a bipartite architecture, composed of a large subunit, hosting the catalytic NiFe(CO)(CN)2 cofactor, and a small subunit whose iron-sulfur clusters are responsible for electron transfer. To analyze in detail the catalytic competence of the large subunit without its smaller counterpart, we purified the large subunit HoxC of the regulatory [NiFe]-hydrogenase of the model H2 oxidizer Ralstonia eutropha to homogeneity. Metal determination and infrared spectroscopy revealed a stoichiometric loading of the metal cofactor. This enabled for the first time the determination of the UV-visible extinction coefficient of the NiFe(CO)(CN)2 cofactor. Moreover, the absence of disturbing iron-sulfur clusters allowed an unbiased look into the low-spin Fe2+ of the active site by Mössbauer spectroscopy. Isolated HoxC was active in catalytic hydrogen-deuterium exchange, demonstrating its capacity to activate H2. Its catalytic activity was drastically lower than that of the bipartite holoenzyme. This was consistent with infrared and electron paramagnetic resonance spectroscopic observations, suggesting that the bridging position between the active site nickel and iron ions is predominantly occupied by water-derived ligands, even under reducing conditions. In fact, the presence of water-derived ligands bound to low-spin Ni2+ was reflected by the absorption bands occurring in the corresponding UV-vis spectra, as revealed by time-dependent density functional theory calculations conducted on appropriate in silico models. Thus, the isolated large subunits indeed represent simple [NiFe]-hydrogenase models, which could serve as blueprints for chemically synthesized mimics. Furthermore, our data point to a fundamental role of the small subunit in preventing water access to the catalytic center, which significantly increases the H2 splitting capacity of the enzyme.

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Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations.

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