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 forma tion and dissociation rate constants k(f) and k(d) of the high-affinit y complex between yeast iso-1-cytochrome c (yCc) and cytochrome c pero xidase compound I (CMPI) over a wide range of ionic strength. These st udies utilized Ru-39-Cc, which contains trisbipyridylruthenium attache d to the cysteine residue in the H39C,C102T variant of yCc, and has th e 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 valu e 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 t o 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 s tep in enzyme turnover is product dissociation below 150 mM ionic stre ngth and intracomplex electron transfer to the oxyferryl heme at highe r ionic strength. The interaction between yCe and CcP is optimized at physiological ionic strength to provide the largest possible complex f ormation rate constant k(f) without allowing product dissociation to b e rate-limiting. The effects of surface mutations on the kinetics prov ided evidence that the high-affinity binding site used for the reactio n in solution is similar to the one identified in the yCc:CcP crystal structure.