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

• Published in: Biochimica et biophysica acta. Bioenergetics 2003-12, Vol.1607 (2), p.65-78
• Main Authors: Ebert, C.Edward, Ghosh, Mousumi, Wang, Yudong, Beattie, Diana S.
• Format: Article
• Language: English
• Subjects: Complex III; Cytochrome bc1 complex; Iron–sulfur protein; Mitochondrial electron transport chain; Proton pumping; Ubiquinol:cytochrome c reductase; Yeast mitochondria
• ISSN: 0005-2728; 1879-2650
• DOI: 10.1016/j.bbabio.2003.08.009

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.