Proton-coupled electron equilibrium in soluble and membrane-bound cytochrome c oxidase from Paracoccus denitrificans.
The pH dependence of electron and proton re-equilibration upon CO photolysis from two-electron-reduced aa3 oxidase was followed by time-resolved electrometry and optical spectroscopy. Optical spectroscopy on soluble Paracoccus denitrificans enzyme at alkaline pH revealed a slow (1 ms) component of electron re-equilibration coupled to the release of protons from the catalytic site. In the work [Brändén, M., et al. (2003) Biochemistry 42, 13178-13184], it was proposed that this proton is released from a water molecule in the catalytic site, located deep in the membrane dielectric. Movement of charged particles such as protons across the dielectric should create an electric potential. However, recording of the time course of the potential generation did not show any potential development in the millisecond time domain, but instead, potential generation was found with an apparent time constant of 50-100 micros. This potential was generated upon proton release from the level of the binuclear catalytic site through the K-channel, because mutation in this channel abolishes the potential generation altogether. The apparent inconsistency between results from optical spectroscopy and electrometry was solved by optical experiments on the membrane-incorporated enzyme. Reconstituting the enzyme into proteoliposomes speeds up the slow electron redistribution process by a factor of 10 and shows the same time constant as potential generation. The possible mechanism of such dramatic change in the rate of proton transfer is discussed.
Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland.
Ilya
I
Belevich
Anne
A
Tuukkanen
Mårten
M
Wikström
Michael I
MI
Verkhovsky
D002248:Carbon Monoxide; D002462:Cell Membrane / Q000201:enzymology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons; D012995:Solubility
D011522:Protons; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV