Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) → 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.

Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and ðNH 4 Þ 2 HPO 4 . The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about −20 kcal∕mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.dimer | trimer | protein polymer

N-terminal truncation of the

No abstract