Ligand mediated reduction of c-type cytochromes by Fe(II): Kinetic and mechanistic insights

Abstract

Cytochrome c (c-Cyts), as a key protein involved in extracellular electron transfer, plays an important role in the microbial Fe(II)-oxidizing process. In this study, due to their similar structure with the outer membrane protein of microorganisms, the equine c-Cyts was employed as a model protein to examine the impact of different organic ligands on the redox kinetics between Fe(II) and c-Cyts using the rapid stopped-flow spectrometer. Our results suggest that the presence of organic ligands will significantly accelerate the reaction between c-Cyts and Fe(II), with reaction rates obtained in the following order: EDTA > citric acid (Cit) > NTA > malonic acid (Mal) > glycine amino acid (Gly) > acetate acid (Ace) > control. It was also found from the kinetic modeling results that the reduction rate of c-Cyts by Fe(II)-ligands was closely correlated to the complex constant between Fe(II) and each ligand, and the reduction rate constants of c-Cyts by Fe(II)L species (L represents “ligands”). Further analysis on the ligand structure indicates that the reaction rate constants are highly correlated with the complex constants of Fe(II)-ligand and the reduction rate constants of c-Cyts by Fe(II)L species (k3) for Group I (Gly, NTA, and EDTA) and Group II (Cit, Mal, and Ace). The results of midpoint potentials of all Fe(II)-ligands obtained through cyclic voltammetry (CV) experiments were ranked as Ace < Gly < NTA < control < Mal < EDTA < Cit, indicating that the redox potentials of Fe(II) complexes in the same group with similar structures were positively correlated with Fe(II)-ligand complex constants. The reorganization energy change of the electron-transfer reaction resulting from the different Fe(II)-ligand structures may contribute significantly more than does the Gibbs free energy (ΔG) to the reaction kinetics of Fe(II)-ligand and c-Cyts.