The aldehyde oxidoreductase from Desulfovibrio gigas belongs to the family of molybdenum hydroxylases. Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S](2+,1+) clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either a FAD group or an electron-transferring protein. When the three metal centers of D. gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin-spin interactions are visible when the EPR spectra are recorded at low temperatures. By studying quantitatively these interactions with a model based on the X-ray crystal structure, which takes into consideration the interactions between the magnetic moments carried by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe-2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal structure, the results provide a detailed description of the electron-transfer system of D. gigas AOR.

Cd and Pb and their sub-cellular distributions were determined in Cu Concentrations of Zn,, composite samples of digestive glands of the common octopus, Octopus vulgaris caught from two areas of the Portuguese coast characterised by contrasting metal contamination. Minor contents of Zn (1%), Cu (2%), Cd (6%) and Pb (7%) were found in the insoluble fraction, consisting of nuclei, mitochondria, lysosomes and microsome operationally separated from the whole digestive gland through a sequential centrifugation. A tendency for linear relationships between metal concentrations in nuclei, mitochondria, lysosomes and whole digestive gland was observed. These relationships suggest that despite low metal content organelles responded to the increasing accumulated metals, which means that detoxifying mechanism in cytosol was incomplete. Poorer correlations between microsome and whole digestive gland did not point to metal toxicity in the analysed compartments. However, the high accumulated Cd indicated that O. vulgaris is an important vehicle of this element to its predators in the coastal environment. (c) 2007 Elsevier B.V. All rights reserved.

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDS/PAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at 4 degrees C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6(1)22 or its enantiomorph, with cell dimensions a = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 x 10(-3) nm3/Da (2.5 A3/Da), consistent with the experimental crystal density, rho = 1.14 g/cm3. One dimer (approximately 2 x 100 kDa) is located on a crystallographic twofold axis.

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P2(1) (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, beta = 106.9 degrees) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, beta = 104.9 degrees) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (lambda = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2(1) data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.