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Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy. [ABSTRACT FROM AUTHOR]

The two resting forms of the active site of [NiFe] hydrogenase, Ni-A and Ni-B, have significantly different activation kinetics, but reveal nearly identical spectroscopic features which suggest the two states exhibit subtle structural differences. Previous studies have indicated that the states differ by the identity of the bridging ligand between Ni and Fe; proposals include OH−, OOH−, H2O, O2−, accompanied by modified cysteine residues. In this study, we use single crystal ENDOR spectroscopy and quantum chemical calculations within the framework of density functional theory (DFT) to calculate vibrational frequencies, 1H and 17O hyperfine coupling constants and g values with the aim to compare these data to experimental results obtained by crystallography, FTIR and EPR/ENDOR spectroscopy. We find that the Ni-A and Ni-B states are constitutional isomers that differ in their fine structural details. Calculated vibrational frequencies for the cyano and carbonyl ligands and 1H and 17O hyperfine coupling constants indicate that the bridging ligand in both Ni-A and Ni-B is indeed an OH− ligand. The difference in the isotropic hyperfine coupling constants of the β-CH2 protons of Cys-549 is sensitive to the orientation of Cys-549; a difference of 0.5 MHz is observed experimentally for Ni-A and 1.9 MHz for Ni-B, which results from a rotation of 7 degrees about the Cα–Cβ–Sγ–Ni dihedral angle. Likewise, the difference of the intermediate g value is correlated with a rotation of Cys-546 of about 10 degrees. [ABSTRACT FROM AUTHOR]

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O 2 -sensitive enzyme that is inactivated in the presence of O 2 but the oxidized enzyme can recover its catalytic activity by reacting with H 2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O 2 , as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17 S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19 S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

[NiFe] hydrogenases catalyze the reversible oxidation of dihydrogen. For this simple reaction the molecule has developed a complex catalytic mechanism, during which the enzyme passes through various redox states. The [NiFe] hydrogenase contains several metal centres, including the bimetallic Ni-Fe active site, iron-sulfur clusters and a Mg(2+) ion. The Ni-Fe active site is located in the inner part of the protein molecule, therefore a number of pathways are involved in the catalytic reaction route. These consist of an electron transfer pathway, a proton transfer pathway and a gas-access channel. Over the last 10-15 years we have been investigating the crystal structures of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F, which is a sulfate-reducing anaerobic bacterium. So far the crystal structures of the oxidized, H(2)-reduced and carbon monoxide inhibited states have been determined at high resolution and have revealed a rather unique structure of the hetero-bimetallic Ni-Fe active site. Furthermore, intensive spectroscopic studies have been performed on the enzyme. Based on the crystal structure, a water-soluble Ni-Ru complex has been synthesized as a functional model for the [NiFe] hydrogenases. The present review gives an overview of the catalytic reaction mechanism of the [NiFe] hydrogenases.