The reactions of hydrogen peroxide with bovine cytochrome c oxidase

Oxidised cytochrome c oxidase is known to react with two molecules of hydro gen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' spec ies. These are widely used as model compounds for the equivalent P and F in termediates of the catalytic cycle. However, kinetic analysis of the reacti on with H2O2 ill the PH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reacti on with a single H2O2. This species, termed F-., is spectrally similar, but not identical, to F. The reactions are equivalent to those previously repo rted for the bo type quinol oxidase from Escherichia coil (T. Brittain, R.H . Little, C. Greenwood, N.J. Watmough, FEES Lett. 399 (1996) 21-25) where i t was proposed that F-. is produced directly from P. However, in the bovine oxidase F-. does not appear in samples of the 607-nm form, P-M, produced b y CO/O-2 treatment, even at low pH, although this form is shown to be ident ical to the H2O2-derived P state, P-H, on the basis of spectral characteris tics and kinetics of reaction with H2O2. Furthermore, lowering the pH of a sample of P-M or P-H generated at high pH results in F-. formation only on a minutes time scale. It is concluded that P and F-. are not in a rapid, pH -dependent equilibrium, but instead are formed by distinct pathways and can not interconvert in a simple manner, and that the crucial difference betwee n them lies in their patterns of protonation. (C) 2000 Elsevier Science B.V . All rights reserved.