An integral approach including experimental and theoretical analysis has been carried out with the wild-type and engineered CODHIICh variant to assess the parameters that control the CN stretching frequency.

[NiFe] hydrogenases are enzymes that catalyze the splitting of molecular hydrogen according to the reaction H2 → 2H+ + 2e–. Most of these enzymes are inhibited even by low traces of O2. However, a special group of O2-tolerant hydrogenases exists. A member of this group is the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha (ReMBH). The ReMBH harbors an unusual iron sulfur cluster with composition 4Fe3S­(6Cys) that is able to undergo structural changes triggering the flow of two electrons to the [NiFe] active site. These electrons promote oxygen reduction at the active site, preventing, in this way, aerobic inactivation of the enzyme. In the superoxidized state, the [4Fe3S] cluster binds to a hydroxyl group that originates from either molecular oxygen or water reaching the site. Both reactions, oxygen reduction to water at the [NiFe]- or [4Fe3S]-centers and oxygen evolution from water at the proximal cluster, require the delivery of protons regulated by a subtle communication mechanism between these metal centers. In this work, we sequentially apply multiscale modeling techniques as quantum mechanical/molecular mechanics methods and classical molecular dynamics simulations to investigate the role of two distinct proton transfer pathways connecting the [NiFe] active site and the [4Fe3S] proximal cluster of ReMBH in the protection mechanism against an oxygen attack. Although the “glutamate” pathway is preferred by protons migrating toward the active site to avoid inactivation by O2, the “histidine” pathway plays an essential role in delivering protons for O2 reduction at the proximal cluster. The results obtained in this work not only provide new pieces to the puzzling catalytic mechanisms governing O2-tolerant hydrogenases but also highlight the relevance of dynamics in the proper description of biochemical reactions in general.

No abstract

The biocatalytic function of carbon monoxide dehydrogenase (CODH) has a high environmental relevance owing to its ability to reduce CO . Despite numerous studies on CODH over the past decades, its catalytic mechanism is not yet fully understood. In the present combined spectroscopic and theoretical study, we report first evidences for a cyanate (NCO ) to cyanide (CN ) reduction at the C-cluster. The adduct remains bound to the catalytic center to form the so-called CN -inhibited state. Notably, this conversion does not occur in crystals of the Carboxydothermus hydrogenoformans CODH enzyme (CODHII ), as indicated by the lack of the corresponding CN stretching mode. The transformation of NCO , which also acts as an inhibitor of the two-electron-reduced C state of CODH, could thus mimic CO turnover and open new perspectives for elucidation of the detailed catalytic mechanism of CODH.

The double-cubane cluster (DCC) refers to an [Fe8S9] iron-sulfur complex that is otherwise only known to exist in nitrogenases. Containing a bridging µ2-S ligand, the DCC in the DCC-containing protein (DCCP) is covalently linked to the protein scaffold via six coordinating cysteine residues. In this study, the nature of spin coupling and the effect of spin states on the cluster’s geometry are investigated computationally. Using density functional theory (DFT) and a broken symmetry (BS) approach to study the electronic ground state of the system, we computed the exchange interaction between the spin-coupled spins of the four FeFe dimers contained in the DCC. This treatment yields results that are in excellent agreement with both computed and experimentally determined exchange parameters for analogously coupled di-iron complexes. Hybrid quantum mechanical (QM)/molecular mechanical (MM) geometry optimizations show that cubane cluster A closest to charged amino acid side chains (Arg312, Glu140, Lys146) is less compact than cluster B, indicating that electrons of the same spin in a charged environment seek maximum separation. Overall, this study provides the community with a fundamental reference for subsequent studies of DCCP, as well as for investigations of other [Fe8S9]-containing enzymes.