Redox Behavior of Heme Embedded in Mycobacterium tuberculosis Bacterioferritin Protein Shell: Impact of Axial Ligands and Non-heme Iron

Abstract

While higher organisms synthesize only heme-free ferritin, pathogenic bacteria such as Mycobacterium tuberculosis (Mtb) express both heme-bound and heme-free analogues. The synthesis of multiple types of ferritin in these bacteria and the roles of heme(s) and conserved axial Met52 in heme-bound bacterioferritin remain a mystery. Though the redox chemistry of heme embedded in bacterioferritin seems crucial, reports on its reduction potential (Em,7) are scarce. Here, the reduction potential and kinetics of heme reduction were obtained to understand its redox behavior in detail. A transient profile for heme reduction and subsequent reoxidation suggests the role of heme as an intrinsic electron relay station in Mtb bacterioferritin. Presence of O2 and non-heme iron impacts heme reduction and detection of transient reduced-heme species. Substitution of axial Met52 affects heme binding and reduction kinetics. Em,7 of heme in wild-type bacterioferritin was found to be unusually negative, which further decreased upon Met52 substitution or incorporation of non-heme iron. These findings, in conjunction with crystallographic analyses, explain the natural preference for Met52. This work establishes the direction of electron flow from reductants to the mineral core via heme and may explain the advantages that bacterioferritin provides to Mtb under limited iron/O2 levels in human macrophages and lungs to meet the iron requirements.