A generalized hybrid orbital (GHO) method has been developed at the semiempirical level in combined quantum mechanical and molecular mechanical (QM/MM) calculations. In this method, a set of hybrid orbitals is placed on the boundary atom between the QM and MM fragments, and one of the hybrid orbitals participates in the SCF calculation for the atoms in the QM region. The GHO method provides a well-defined potential energy surface for a hybrid QM/MM system and is a significant improvement over the “link-atom” approach by saturating the QM valencies with hydrogen atoms. The method has been tested on small molecules and yields reasonable structural, energetic, and electronic results in comparison with the results of the corresponding QM and MM approximations. The GHO method will greatly increase the applicability of combined QM/MM methods to systems comprising large molecules, such as proteins.

The 2.54 A resolution structure of Ni-Fe hydrogenase has revealed the existence of hydrophobic channels connecting the molecular surface to the active site. A crystallographic analysis of xenon binding together with molecular dynamics simulations of xenon and H2 diffusion in the enzyme interior suggest that these channels serve as pathways for gas access to the active site.

The crystal structure of the membrane-bound O 2 -tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H 2 -reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O 2 -sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oϵ of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H 2 -reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O 2 to H 2 O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O 2 reduction, located near the active site.[4Fe-3S] cluster | membrane-bound hydrogenase | Mössbauer spectroscopy | QM/MM | structure/function relationships

Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro)peroxo intermediates with the (hydro)peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron-oxygen bond in order to release the reaction product, hydrogen peroxide.

؊ with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.

Reactions involving H(2), N(2), CO, CO(2) and CH(4) are likely to have been central to the origin of life. This is indicated by the active-site structures of the enzymes involved, which are often reminiscent of minerals. Through the combined efforts of protein crystallography, various types of spectroscopy, theoretical calculations and model chemistry, it has been possible to put forward plausible mechanisms for gas-based metabolism by extant microorganisms. Although the reactions are based on metal centres, the protein matrix regulates reactivity and substrate and product trafficking through internal pathways, specific ligation and dielectricity.

The center of attention: IscS cysteine desulfurases and IscU scaffolds are involved in biological iron–sulfur cluster assembly. The X‐ray structure of an anaerobically produced, mutated (Fe2S2‐(IscS‐IscUD35A))2 complex reveals a cluster coordinated by three IscU cysteines and the IscS active cysteine (see picture). In air‐exposed crystals the cluster is oxidized to an Fe2S–S center; D35 is essential for complex dissociation.

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