Aspartate-186 in the head group of the yeast iron-sulfur protein of the cytochrome bc1 complex contributes to the protein conformation required for efficient electron transfer.
Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron-sulfur protein of the cytochrome bc(1) complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively. The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron-sulfur protein in the complex. Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron-sulfur protein or the [2Fe-2S] cluster. The midpoint potential of the [2Fe-2S] cluster of the D186A mutant was decreased from 281 to 178 mV. The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron-sulfur protein; however, the redox potential of the [2Fe-2S] cluster of D186N was decreased from 281 to 241 mV. Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron-sulfur protein resulting in changes in the orientation of the [2Fe-2S] cluster and consequently a lowered redox potential. The rate of electrogenic proton pumping in the bc(1) complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H(+)/2e(-) ratio of 1.9 was identical in the mutant and the wild-type complexes. The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc(1) complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H(+)/2e(-) ratio to 1.2 and 1.1, respectively. EEDQ was bound to the iron-sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron-sulfur protein is not required for proton translocation in the bc(1) complex.
Ebert
C Edward
CE
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, P.O. Box 9142, Morgantown, WV 26506-9142, USA.
Ghosh
Mousumi
M
Wang
Yudong
Y
Beattie
Diana S
DS
D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology*; D014450:Electron Transport Complex III / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015003:Yeasts / Q000378:metabolism*
D001224:Aspartic Acid; D001120:Arginine; D014450:Electron Transport Complex III