Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement.
The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.
Mus-Veteau
I
I
Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique (CNRS) BP71, Marseille, France.
Dolla
A
A
Guerlesquin
F
F
Payan
F
F
Czjzek
M
M
Haser
R
R
Bianco
P
P
Haladjian
J
J
Rapp-Giles
B J
BJ
Wall
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016969:Desulfovibrio vulgaris / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006418:Heme / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D015183:Restriction Mapping
D003574:Cytochrome c Group; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D006418:Heme; C017165:cytochrome c(3)