An electrochemical and molecular dynamics investigation of ligand exchange in cytochrome c: Cyanide, histidine and methionine as ligands

The rate of dissociation, kf,AC, of cyanide from the oxidized cyano-cytochrome c complex was determined using normal pulse voltammetry. The rate was 2.94 x 10-4s-1. The rate of association of the oxidized cytochrome c and cyanide was calculated to be 1.35 x 10-3 M-1s-1. These results are in agreement with the known theory about cytochrome c and exogenous ligands in neutral or alkaline solution. It is known that in the oxidized state, cytochrome c prefers to combine with cyanide rather than its 6th axial ligand, Methionine-80. This implies that the rate of association should be faster than the rate of dissociation in the ligand exchange reaction. kf,AC was determined in the iso-1-cytochrome c, variant, Phenylalanine-82-Histidine (Phe82His) and it was ten times faster than what was obtained in the cytochrome c cyanide-system. In the cytochrome c-CN system, the cyanide ion was electrostatically bound to the Fe3+. Hence it was more difficult for the cyanide to leave the complex than it was for Histidine-82, which was covalently bound to the Fe3+. Molecular dynamics study on Phe82His could not show why in cyclic voltammetric experiment, the peak corresponding to the re-oxidation of His82-Fe2+ -His18 was barely seen, no matter the scan rate.;Met80Ala/Phe82His variant was studied using square-wave voltammetry. A peak with a formal reduction potential of -115 mV versus SHE was obtained. This peak was assigned to the coordination due to Histidine-82.;Cyclic and square-wave voltammetric studies of mouse testicular cytochrome c (MTC) and mouse cytochrome c (MC) were performed. The formal reduction potential of MTC and MC were obtained as 241 and 262 mV vs SHE respectively. The formal reduction potential of MTC being lower suggests that it is a better reducing agent. This could explain why MTC catalyzes the destruction of reactive oxygen species in the mitochondria much more faster than MC.;The Molecular dynamics simulations on the protein variants, Threonine-78-Serine (Thr78Ser) and Threonine-78-Valine (Thr78Val) indicated that the structural water, water 166, was lost when Threonine was replaced with Valine, but it was retained when Threonine was replaced with Serine. This observation supports an earlier work with square-wave voltammetry which stipulated that mutation of cytochrome c in which Threonine was replaced with Serine did not cause any appreciable changes to the electrochemical properties of the cytochrome.