The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition.
The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 microM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 microM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.
Thönström
P E
PE
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Nilsson
T
T
Malmström
B G
BG
D000818:Animals; D002417:Cattle; D003486:Cyanides / Q000494:pharmacology*; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000494:pharmacology*; D011522:Protons; D013053:Spectrophotometry
D003486:Cyanides; D011522:Protons; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide