Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity.
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.
Cocco
T
T
Institute of Medical Biochemistry and Chemistry, Consiglio Nazionale delle Ricerche, University of Bari, Italy.
Di Paola
M
M
Papa
S
S
Lorusso
M
M
D000818:Animals; D002417:Cattle; D014450:Electron Transport Complex III / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D017494:Proton Pumps / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D017494:Proton Pumps; D014450:Electron Transport Complex III