Chemical Modification of the Bovine Mitochondrial bc1 Complex Reveals Critical Acidic Residues Involved in the Proton Pumping Activity

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Abstract
Bovine heart ubiquinol−cytochrome c reductase (bc1 complex) was modified with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), which is a selective reagent for buried carboxyl groups. EEDQ treatment caused a loss of the proton pumping activity of liposome-reconstituted bc1 complex, without effect on the passive proton conductivity of the proteoliposomes. Although the decoupling effect produced on proton translocation was similar to that elicited by N,N‘-dicyclohexylcarbodiimide (DCCD) modification of cytochrome b and subunit IX, EEDQ modified different subunits, namely the Core protein II and the iron−sulfur protein (ISP). A time-dependent increase of the labeling of both subunits was observed which was kinetically comparable with the decrease of the H+/e- ratio. Trypsin treatment of the complex showed that the EEDQ-modified carboxyl group in the ISP belongs to the protruding moiety of the protein, holding the Fe/S cluster. The results obtained show that critical acidic residues, located in different subunits of the bc1 complex, at both sides of the membrane, contribute to its proton pumping activity.

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