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Hydrogenases are abundant enzymes that catalyse the reversible interconversion of [H.sub.2] into protons and electrons at high rates (1). Those hydrogenases maintaining their activity in the presence of [O.sub.2] are considered to be central to [H.sub.2]-based technologies, such as enzymatic fuel cells and for light-driven [H.sub.2] production (2). Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations (3-8), the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by [O.sub.2] has remained unclear. Here we present the crystal structure of an [O.sub.2]-tolerant [NiFe]-hydrogenase from the aerobic [H.sub.2] oxidizer Ralstonia eutropha H16 at 1.5 A resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the [H.sub.2]-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of [H.sub.2] oxidation and as an electron-delivering device upon [O.sub.2] attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials (7-9). The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical [H.sub.2]-converting catalysts that are capable of cycling [H.sub.2] in air., More than two billion years ago, ancient microbes exploited the reducing power of [H.sub.2] for their metabolism; until today [H.sub.2] provides a valuable energy source which is used by [H.sub.2]-oxidizing [...]

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2tolerance of the enzyme. [ABSTRACT FROM AUTHOR]

Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H2, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry--that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H2 oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O2, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N10-formyl-tetrahydrofolate (N10-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N10-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates. [ABSTRACT FROM AUTHOR]

Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coil Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp 164 (by Asn) and Lys167 (by Arg or Gin) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition. [ABSTRACT FROM AUTHOR]

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O 2 -tolerant hydrogenase. It catalyzes the oxidation of H 2 into 2e - and 2H + in the presence of high O 2 concentrations. This characteristic trait is intimately linked to the unique Cys 6 [4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys 5 [4Fe-4S] or Cys 4 [4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O 2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O 2 protection mechanism that detoxifies O 2 to H 2 O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O 2 binding under electron-deficient conditions., Competing Interests: The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P. S. (patrick.scheerer@charite.de), O. L. (oliver.lenz@tu-berlin.de), and I. Z. (ingo.zebger@tu-berlin.de)., (This journal is © The Royal Society of Chemistry.)

This corrects the article DOI: 10.1038/srep44628.