Cytochrome c is an important heme protein in the electron transport chain that transfers electrons between Complexes III (coenzyme Q – cyt c reductase) and IV (cyt c oxidase) as its heme converts between ferrous and ferric states. An important property of wild-type cytochrome c is the ability of a lysine side chain to displace methionine-80 as a ligand to the ferric heme at high pH. This "alkaline transition" has been linked to the signaling pathway for programmed cell death in eukaryotic cells. A cytochrome c mutant, T49V/T78V/K79G, was evaluated using cyclic voltammetry (CV) and square wave voltammetry (SWV) at pH 7.4 to determine the effect that these substitutions in the hydrogen-bonding network at the heme have on reduction potential and ligation properties of the heme iron. Voltammetry can follow the ligand exchange since the lysine-bound state has a substantially lower reduction potential than the methionine-bound form. Both CV and SWV show that in the ferric form this mutant highly favors the Lys-ligated species over the Met-ligated species, even at neutral pH. Rapidly oxidizing and reducing the protein by increasing the potential scan rate shows that ligand exchange upon oxidation occurs extremely rapidly, faster than what has previously been observed for variants that maintain either or both threonine-49 and threonine-78. Indeed, the ligand exchange from methionine to lysine upon oxidation is so rapid and complete that unambiguous signals attributable to the ferric Met-bound protein were not readily observed. However, by chemically reducing the sample with dithionite solution and using large potential jumps, SWV reveals that the ferric Met-bound form can be accessed transiently.

Rapid ligand exchange in a cytochrome c mutant

ACS Spring 2022

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