Reduction of c-type cytochromes by Fe(II)-ligand under oxic conditions: Roles of Fe(II)-heme complexation and reactive oxygen species

Abstract

Microbial Fe(II) oxidation is mainly mediated by the outer-membrane proteins that contain c-type cytochromes (c-Cyts), and the widespread Fe(II)-oxidizing bacteria are usually subjected to fluctuations between oxic and anoxic conditions. However, it remains unclear how oxygen affects the redox dynamics of c-Cyts by Fe(II), particularly in the presence of organic ligands that are widely distributed in the environment. Here, the reduction of a model heme-containing c-Cyts by Fe(II) was investigated in the absence and presence of organic ligands (i.e., citrate and acetate) at pH 5.5 under anoxic and oxic conditions. The kinetic results showed that c-Cyts reduction was enhanced by citrate and acetate under anoxic conditions, and the enhancement by citrate was more pronounced than that by acetate. The speciation calculation and cyclic voltammetry results suggested that the proportions of Fe(II)-Citrate complexes were much higher than those of Fe(II)-Acetate complex, but the addition of acetate and citrate caused minor changes in redox potential of Fe(II)/Fe(III) species. Further analysis using quantum chemical computation demonstrated that the formation of Fe(II)-Heme complexes via lateral binding to the carboxyl group of c-Cyts was more stable than those via replacing the S-linked axial ligand of heme, with the recombination energy with Heme ranking as Fe(II)-Citrate < Fe(II)-Acetate < Fe(II). Therefore, the stronger complexation of Fe(II)-Citrate and its lower recombination energy with c-Cyts drive the enhanced c-Cyts reduction under anoxic conditions. By contrast, the c-Cyts reduction by Fe(II) and Fe(II)-ligand was inhibited under oxic conditions. The electron spin resonance characterization indicated that both O2−• and OH• radicals were produced in the system of c-Cyts with Fe(II) or Fe(II)-ligand under oxic conditions and the signals were higher with Fe(II)-ligand than those with Fe(II). These reactive oxygen species were proposed to enable a re-oxidation of the c-Cyts that was reduced by Fe(II) or Fe(II)-ligand, causing an inhibitory effect on the observed c-Cyts reduction and forming more reactive oxygen species. Our findings shed light on the important roles of Fe(II)-ligand-heme complexation and reactive oxygen species in the interaction between Fe(II) and c-Cyts, which deepen the understanding of microbial/enzymatic Fe(II) oxidation under oxic conditions at the molecular level.