Crystal structure of cytochrome c3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.

International audienceThe crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki.The crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki

Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I fromDesulfovibrio desulfuricans (Norway strain)

International audienceA three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfatereducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the “best” structure in terms of charges, interactions, and complementary f the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 Å, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 Å2

Extensive 1H-NMR resonance assignment of proteins using natural abundance gradient-enhanced 13C-1H correlation spectroscopy.

AbstractThe reliability and completeness of 1H NMR resonance assignment can be improved by the use of 13C−1H HSQC correlation spectra on unlabelled protein samples using pulsed field gradients. This technique is illustrated on a 5.2 mM sample of the 79 residue Desulfovibrio vulgaris ferrocytochrome c553. Protons attached to the same carbon can be unambiguously paired in a HSQC spectrum. Contrary to 1H, most amino acids exhibit characteristic 13C chemical shift ranges, which can be used for 13C assignment. This technique is especially useful for long side chain residues, such as Gln, Glu, Lys, Arg

Intramolecular electron transfer in ferredoxin II from Desulfovibrio desulfuricans Norway.

International audienceIn order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms.In order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms

Crysrallization and preliminary crystallographic study of an octaheme cytochrome c3 from Desulfovibrio desulfuricans Norway.

International audienceAn octa-heme cytochrome c3, isolated as a dimeric molecule of about 30 kDa from the anaerobic bacteria Desulfovibro desulfuricans Norway, has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals are trigonal, space group P3(1)21 (or its enantiomorph P3(2)21), with cell dimensions: a = b = 72.9 A c = 62.7 A. The asymmetric unit contains most probably one monomer and a solvent content of about 60%. Under this assumption, the crystallographic 2-fold axis relates the two subunits of the dimer. Diffraction extends to 2.0 A.An octa-heme cytochrome c3, isolated as a dimeric molecule of about 30 kDa from the anaerobic bacteria Desulfovibro desulfuricans Norway, has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals are trigonal, space group P3(1)21 (or its enantiomorph P3(2)21), with cell dimensions: a = b = 72.9 A c = 62.7 A. The asymmetric unit contains most probably one monomer and a solvent content of about 60%. Under this assumption, the crystallographic 2-fold axis relates the two subunits of the dimer. Diffraction extends to 2.0 A

Interaction studies between redox proteins, cytochrome c3 , ferredoxin and hydrogenase from sulfate reducing bacteria.

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