Ionic strength effects on cytochrome aa3 kinetics

Abstract

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa 3 can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa 3 increases, whereas in the lower ionic strength region the formation of a less active cytochrome c- aa 3 complex limits the ferrocytochrome c association to the low affinity site. 2. At low ionic strength, the reduction of cytochrome c- aa 3 complex by ferrocytochrome c 1 proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c 1 and aa 3. 3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c- aa 3 complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c. 4. Dissociation rates of both high and low affinity sites on cytochrome aa 3 were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 μM for the high and low affinity site, respectively.