Kinetics of ligand-binding and oxidation-reduction reactions of cytochrome c from horse heart and Candida krusei.

Reactions of cytochromes c from horse heart and Candida krusei (a yeast) were studied at 25” under pseudo-first order conditions by a flow technique. The rate law for the reduction of C. krusei by Cr(I1) (pH 6.0, p = 1.0 M) in chloride is kobs = a[Cr(II)]/(l + b[Cr(II)]), where a and b are 1.0 x 10’ M-’ s-l and 167 f 38 ~-l, respectively. The binding of imidazole (Im) to C. krusei conforms to the rate law k&s = k, + R,[Im] (k, = 1.65 f 0.15 s-l, 4, = 16.4 f 0.3 I@ s-l at 695 nm, pH 7.1, p = 1.00 M) which is consistent with the kf reaction C. krusei + Im ,c C. krusei-Im. Under the k, same conditions the equilibrium constant for formation of the C. krusei-imidazole complex determined from absorbance changes is 11.0 f 0.6 10 which, within experimental error, is identical with the ratio k,/k, = 9.9 f 0.9 16. For reduction of ferricytochrome c by pentaamminebenzimidazoleruthenium(I1) (Ru(I1)) and oxidation of ferrocytochrome c by ferricyanide (Fe(IlI)), the rate laws are of the form kobs = k[X], which is consistent with the simple process, k cyt+xproducts. For C. krusei and horse heart, respectively, the rate constants (k) measured are as follows: X = Ru(II), k = (1.0 f 0.3) x lo6 M-’ s-‘, 4.7 x 1Oj M-’ S-’ (pH 6.1, p = 1.00 M); X = Fe(III), k = (2.1 f 0.2) X lo7 M-I s-l, 1.2 x 10’ M-I s-l (pH 7.2, p = 0.10 M). The rate law found for the reduction of cyanoferricytochrome c by dithionite (pH 6.4, /* = 1.00 M) is k,,bs = k’[S2042-]1/2, where k’ = 9.2 M-‘l2 s-l (C. krusei) and 25.9 M-‘12 s-l (horse heart). This rate law is consistent with a rate-determining reduction by SOzformed in a rapidly established preequilibrium dissociation of SIO1*into SOzradicals. The immediate products of the dithionite reduction of the cyanoderivatives are the cyanoferrocytochromes c. Their conversion to the native ferrocytochromes c, monitored by conventional techniques, was found to be a first order process (pH 6.4, p = 1.0 M) with rate constants 6.8 X lOma s-l (C. krusei) and 5.0 x lOma s-l (horse heart). As might have been predicted from their structural similarities, the cytochromes from the two species exhibit no major reactivity differences.