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

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2 e– and 2 H+ 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. Herein, 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.

Sigmatropic rearrangements constitute an important group of pericyclic reactions.I nc ontrast to cycloaddition reactions,e xamples of catalytic variants of electrocyclic reactions and sigmatropic rearrangements are still scarce in the chemical literature.H erein, we report the first organocatalytic Cope rearrangement of in situ-generated divinylcyclopropanes.The reactive motif was generated by condensation of 4-(2-vinylcyclopropyl)but-2-enal derivatives with as econdary amine catalyst to form at ransient dienamine.T he cycloheptadiene products could be obtained in high yield and excellent diastereoselectivity.I mportantly,t he reaction was demonstrated to be stereospecific,p roceeding under mild conditions,while exhibiting broad functional group tolerance.

[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.

No abstract