The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

Abstract

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH ∗ 7.0) the electron self-exchange rate constants, k ex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 × 10 3 and 6 × 10 4 M −1·s −1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron selfexchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN) 6 3− catalyse electron self-exchange. At saturation a limiting value of ~ 1.4 × 10 5 M −1·s −1 is observed for both anions.