The effects of pH and ionic strength on cytochrome c oxidase steady-state kinetics reveal a catalytic and a non-catalytic interaction domain for cytochrome c.

The influence of pH and ionic strength on the steady-state kinetics of purified bovine cytochrome c oxidase was studied by spectrophotometry. At low ionic strength, increasing the pH in the range between 5.4 and 8.6 resulted in a slight decrease in maximal turnover numbers of the high-affinity and the low-affinity reactions. The high-affinity Km was also found to decrease with increasing pH. The ionic-strength dependence of the steady-state kinetics of positively charged cytochrome c oxidase at pH 6.2 and that of negatively charged cytochrome c oxidase at pH 7.8 were similar; in both cases, high-affinity Km values and high-affinity and low-affinity TNmax values increased with ionic strength. The low-affinity Km was independent of both pH and ionic strength. Above I = 100 mM, no low-affinity reaction could be observed. A description of the electrostatic interactions between cytochrome c and cytochrome c oxidase, based on the overall monopoles and overall dipoles of the two proteins, could not explain our data. We propose that at I greater than or equal to 25 mM such an approximation cannot be used for electrostatic interactions between large proteins, since the assumption that all charges on the surfaces of the reacting proteins would contribute equally to the electrostatic interaction is not valid. A qualitative description of electrostatic interactions between the two cytochromes based on limited electrostatic interaction domains on the cytochrome c oxidase surface was found to be in good agreement with all our data and supports the model of Speck et al. (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 347-351), who proposed one catalytic and one non-catalytic cytochrome c binding site. It is proposed that the allosteric effect of the cytochrome c at the non-catalytic site is of an electrostatic nature. At high ionic strength (occurring in vivo), this cytochrome c molecule would then no longer affect the catalytic site, resulting in the absence of the low-affinity reaction.