CO binding to mitochondrial mixed valence state cytochrome oxidase at low temperatures

Abstract

The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178–203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444–463 nm in the Soret region, and 590–630 and 608–630 nm in the α region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem. J. 175, 709–725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beer's law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species II MC) yields unliganded mixed valence state cytochrome oxidase (species E M) and free CO which then recombine to form species I MC; species I MC is then converted into species II MC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709–725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state ( a 2+ 3Cu + B · a 3+Cu 2+ A) and fully reduced ( a 2+ 3Cu + B · a 2+Cu + A) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and Cu A has a significant effect on the CO binding properties of cytochrome a 3 and possibly Cu B.