Characterization and oxidoreduction properties of cytochrome c3 after heme axial ligand replacements.
Cytochrome c3 (M(r) 13,000) is a tetrahemic cytochrome in which the four heme iron atoms are coordinated by 2 histidine residues at the axial positions. The presence of several oxidoreduction centers in the same molecule raises the question of their coupling. To investigate this mechanism, four single mutations were introduced in cytochrome c3 by site-directed mutagenesis, leading to the replacement of each histidine, the sixth axial ligand of the heme iron atom, by a methionine residue. Characterization of the new set of molecules using biochemical and biophysical techniques was carried out. The novel methionine was correctly coordinated to the iron atom of hemes 3 and 4 in H25M and H70M cytochromes c3, respectively, and this coordination induced a large increase in the oxidoreduction potential of the mutated heme. In contrast, in the case of H22M and H35M cytochromes c3, in which the corresponding methionine is in an oxidized form, only slight changes in redox potential values were observed. In H22M, H25M, and H35M cytochromes c3, two conformations of the molecule were possible, in which the methionine is either free or coordinated to the iron atom. The rate constants for the electron exchange reactions between the cytochrome mutants and the hydrogenase were measured using electrochemical techniques. Distinct behaviors were revealed depending on the mutation. The values of the rate constants for the electron exchange reactions are interpreted in terms of intramolecular electron exchange among the four hemes of the cytochrome.
Dolla
A
A
Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique, Marseille, France.
Florens
L
L
Bianco
P
P
Haladjian
J
J
Voordouw
G
G
Forest
E
E
Wall
J
J
Guerlesquin
F
F
Bruschi
M
M
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003901:Desulfovibrio / Q000235:genetics / Q000378:metabolism*; D016969:Desulfovibrio vulgaris / Q000378:metabolism; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; C017165:cytochrome c(3)