Transmembrane charge separation during the ferryl-oxo -> oxidized transition in a nonpumping mutant of cytochrome c oxidase.
The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).
Siletsky
Sergey A
SA
AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119 992, Russia.
Pawate
Ashtamurthy S
AS
Weiss
Kara
K
Gennis
Robert B
RB
Konstantinov
Alexander A
AA
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017666:Deuterium Oxide; D004563:Electrochemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D017494:Proton Pumps; D011522:Protons; D006418:Heme; D003576:Electron Transport Complex IV; D017666:Deuterium Oxide