Site Directed Mutagenesis of the Cyc2 Outer Membrane Protein from Acidithiobacillus ferrooxidans Reveals a Critical Role for Bound Iron Atoms in Extracellular Electron Transfer

Abstract

Extracellular electron transfer (EET) processes by metal respiratory bacteria rely on outer membrane proteins (OMPs) to exchange electrons across the insulating cell membrane. The most studied OMPs from metal reducing bacteria contain multiple sequential heme groups. However, many iron‐oxidizing bacteria, including the industrial bioleaching microbe Acidithiobacillus ferrooxidans, contain monoheme OMPs and the mechanism of electron transfer through these smaller structures has not been elucidated. Computational modeling was previously used to predict two iron ion binding sites in the Cyc2 protein structure from A. ferrooxidans. To determine if these binding sites are critical for protein function, the monoheme Cyc2 OMP from A. ferrooxidans is recombinantly expressed in E. coli outer membrane vesicles (OMVs) which are then incorporated into biomimetic cell‐membrane supported lipid bilayers (SLB) on electrodes to measure electron transfer. Site‐directed mutagenesis is used to disrupt the putative ion binding sites predicted from modeling to elucidate the mechanism. It is confirmed that the Cyc2 protein is capable of EET without the need for soluble iron or other accessory proteins. These results confirm the critical role of bound metal ions in the A. ferrooxidans EET mechanism, and it is expected that homologous monoheme OMPs will have similar conduction pathways.