Evidence for quinol oxidase activity of ImoA, a novel NapC/NirT family protein from the neutrophilic Fe(II) oxidizing bacterium Sideroxydans lithotrophicus ES-1

Abstract

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1.