Kinetics of Ligand-binding and Oxidation-Reduction Reactions of Cytochrome c from Horse Heart and Candida krusei

Abstract

Abstract 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(II) (pH 6.0, µ = 1.0 m) in chloride is kobs = a[Cr(II)]/(l + b[Cr(II)]), where a and b are 1.0 x 104 m-1 s-1 and 167 ± 38 m-1, respectively. The binding of imidazole (Im) to C. krusei conforms to the rate law kobs = kr + kf[Im] (kr = 1.65 ± 0.15 s-1, kf = 16.4 ± 0.3 m-1 s-1 at 695 nm, pH 7.1, µ = 1.00 m) which is consistent with the reaction C. krusei + Im (kf)/rlarr2;/(k↑) C. krusei-Im. Under the same conditions the equilibrium constant for formation of the C. krusei-imidazole complex determined from absorbance changes is 11.0 ± 0.6 m-1 which, within experimental error, is identical with the ratio kf/kr = 9.9 ± 0.9 m-1. For reduction of ferricytochrome c by pentaamminebenzimidazoleruthenium(II) (Ru(II)) and oxidation of ferrocytochrome c by ferricyanide (Fe(III)), the rate laws are of the form kobs = k[X], which is consistent with the simple process, cyt + X (k)/→ products. For C. krusei and horse heart, respectively, the rate constants (k) measured are as follows: X = Ru(II), k = (1.0 ± 0.3) x 106 m-1 s-1, 4.7 x 1O5 m-1 s-1 (pH 6.1, µ = 1.00 m); X = Fe(III), k = (2.1 ± 0.2) x 107 m-1 s-1, 1.2 x 107 m-1 s-1 (pH 7.2, µ = 0.10 m). The rate law found for the reduction of cyanoferricytochrome c by dithionite (pH 6.4, µ = 1.00 m) is kobs = k'[S2O42-]1/2, where k' = 9.2 m-1/2 s-1 (C. krusei) and 25.9 m-1/2 s-1 (horse heart). This rate law is consistent with a rate-determining reduction by SO2- formed in a rapidly established preequilibrium dissociation of S2O42- into SO2- radicals. 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, µ = 1.0 m) with rate constants 6.8 x 10-3 s-1 (C. krusei) and 5.0 x 10-3 s-1 (horse heart). As might have been predicted from their structural similarities, the cytochromes from the two species exhibit no major reactivity differences.