Redox dynamics and equilibria of c-type cytochromes in the presence of Fe(II) under anoxic conditions: Insights into enzymatic iron oxidation

Although c-type cytochrome (c-Cyt) involvement in many biological Fe(II) oxidation processes under microaerobic or anoxic conditions has been proposed, the detailed understanding of the molecular redox reaction of c-Cyts in the presence of Fe2 + is still lacking. In this study, the in situ spectral kinetics and spectroelectrochemistry of a commercial c-Cyt as a model protein was investigated under various environmental conditions (Fe(II) concentration, pH, and ionic strength). The concentrations of reduced c-Cyt (c-Cytred) increased gradually over time and then reached a steady state. The c-Cytred generation rate constants (k) increased with increasing initial Fe(II) concentrations (5–1000 μM), because the redox potentials (Eh) of Fe(II)/Fe(III) were lower with higher Fe(II) concentrations. The k values for c-Cytred generation increased with pH (5.5–8.0). The Eh of Fe(II)/Fe(III) decreased substantially with a slope of 0.177 per pH unit, revealing that the reduction capacity of Fe2 + increased dramatically with pH elevation. In addition, spectroelectrochemistry results revealed that the extent of c-Cytred was strongly dependent on pH, indicating that H+ was involved in the c-Cyt reduction process. Hence, the pH effects were attributed to the involvements of H+ in the reactions between Fe2 + and c-Cyt. The k values for c-Cytred generation decreased with increasing NaCl concentrations (0–1.0 M). The correlations between the kinetic rates and the concentrations/activities of Fe(II) species are discussed, with the results demonstrating that Fe2 + activity was the major factor influencing the reaction kinetics with different ionic strength. This study provides insights into the mechanisms of enzymatic iron oxidation process under anoxic conditions at the molecular level.