Role of acidic and aromatic amino acids in Rhodobacter capsulatus cytochrome c1. A site-directed mutagenesis study.
The roles of two evolutionarily conserved aromatic residues in the cytochrome c(1) component of the Rhodobacter capsulatus cytochrome bc(1) complex, phenylalanine 138 and tyrosine 194, were analyzed by site-directed mutagenesis, in combination with biophysical and biochemical measurements. Changing Phe138 to either alanine or valine, but not to tyrosine, results in redox heterogeneity of cytochrome c(1). Replacement of Phe138 by an aliphatic amino acid also caused changes in the EPR spectrum of the cytochrome and resulted in decreases in the steady-state V(max) for the hydroquinone/cytochrome c oxidoreductase activity of cytochrome bc(1) complexes containing the mutated cytochrome c(1). These findings indicate that the presence of an aromatic residue at position 138 is essential for maintaining the native environment of the cytochrome c(1) heme. In contrast, replacement of Tyr194 by aliphatic amino acids had no significant effect on either the E(m) of cytochrome c(1) or the steady-state activity parameters. Site-directed mutagenesis of glutamate and aspartate residues in a conserved acidic patch (region 2) on Rb. capsulatus cytochrome c(1) suggests that these negatively charged residues do not play a role in the docking of cytochrome c(2) with the cytochrome bc(1) complex.
Li
Jun
J
Department of Chemistry and Biochemistry and Center for Biotechnology and Genomics, Texas Tech University, Lubbock, Texas 79401-1061, USA.
Osyczka
Artur
A
Conover
Richard C
RC
Johnson
Michael K
MK
Qin
Hong
H
Daldal
Fevzi
F
Knaff
David B
DB
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002645:Chickens; D003575:Cytochromes c1 / Q000737:chemistry* / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry; D010788:Photosynthesis; D011485:Protein Binding; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry; D015003:Yeasts / Q000378:metabolism
D000596:Amino Acids; D014443:Tyrosine; D006418:Heme; D010649:Phenylalanine; D003575:Cytochromes c1