Exploring the proton pump mechanism of cytochrome c oxidase in real time.
Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms. This remarkable membrane-bound enzyme also converts free energy from O(2) reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump. Although the structures of several oxidases are known, the molecular mechanism of redox-linked proton translocation has remained elusive. Here, correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques after laser-activated electron injection into the oxidized enzyme. The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties. The 10-micros electron transfer to heme a raises the pK(a) of a "pump site," which is loaded by a proton from the inside of the membrane in 150 micros. This loading increases the redox potentials of both hemes a and a(3), which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a(3)/Cu(B) center, and the electron is transferred to Cu(B). Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.
Belevich
Ilya
I
Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland.
Bloch
Dmitry A
DA
Belevich
Nikolai
N
Wikström
Mårten
M
Verkhovsky
Michael I
MI
D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D004583:Electrons; D007700:Kinetics; D010231:Paracoccus denitrificans / Q000201:enzymology*; D017494:Proton Pumps / Q000378:metabolism*; D011522:Protons; D055672:Static Electricity; D013997:Time Factors
D017494:Proton Pumps; D011522:Protons; D003576:Electron Transport Complex IV