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

Abstract

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.