Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site

A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants k(f) and k(d) of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C,C102T variant of yCc, and has the same reactivity with CMPI as native yCc. k(d) and k(f) were measured by photoreducing a small concentration of Ru-39-Ce in the presence of the oxidized yCc(III):CMPI:CMPI complex, which must dissociate before Ru-39-Cc(II) can bind to CMPI and reduce the radical cation, The value of k(d) for the 1:1 high-affinity complex is very small at low ionic strength, <5 s(-1) but is increased significantly by binding yCe to a second low-affinity site. However, the low-affinity yCe binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value of k(d) increases to 4000 s(-1) at 150 mM ionic strength, while k(f) decreases from >3 x 10(9) M(-1) s(-1) at low ionic strength to 1.3 x 10(9) M(-1) s(-1) at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCe and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant k(f) without allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.