Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus.
Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.
Gray
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Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
Davidson
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Daldal
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002248:Carbon Monoxide / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003573:Cytochrome b Group / Q000378:metabolism; D003575:Cytochromes c1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D015183:Restriction Mapping; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D013053:Spectrophotometry
D003573:Cytochrome b Group; D006418:Heme; D002248:Carbon Monoxide; D003575:Cytochromes c1; D008715:Methionine; D007501:Iron