Effects of the Tyr-67 acetylation on the heme crevice structure of horse heart cytochrome c derivatives

Crystal structures of several cytochromes c indicate that the phenolic group of Tyr-67 (or its homologue) participates in hydrogen bonding interactions within the heme crevice that are significantly affected by oxidation/reduction of the cytochrome. Tyr-67 of N-epsilon-guanidinated cytochrome c (Guan cyt c) and N-epsilon-acetimidylated cytochrome c (Acim cyt c) can be specifically and quantitatively O-acetylated with 3mM N-acetylimidazole. The Tyr-67-acetylated derivatives of Guan cyt c and Acim cyt c (Ac-Guan cyt c and Ac-Acim cyt c) retain the 695 nm absorption band, have a weak negative Cotton effect at 695 nm in their circular dichroism spectra and have almost full activity in the mitochrondrial succinate oxidase system. Cyanide (CN)-, azide-, and imidazole-binding studies were done to evaluate heme ligand stability. Dissociation constants of complexes of the acetylated derivatives are smaller by 10X and 5X than those of cyt c and the unacetylated derivatives, respectively. Pseudofirst order rate constants for CN-binding by Ac-Guan cyt c are 10X and 5X greater than those obtained with cyt c and Guan cyt c. 1H-NMR was used to evaluate structural changes accompanying Tyr-67 acetylation. The most significant spectral effects of acetylation are +6 ppm and +3 ppm changes in the chemical shifts of the resonances arising from the axial methionine-epsilon-methyl protons and the heme c ring methyl-5 protons, respectively. Acetylation of Tyr-67 appears to alter the position of Met-80 relative to the heme ring