Identification and characterization of MtoA: a decaheme c-type cytochrome of the neutrophilic Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1

The Gram-negative bacterium Sideroxydans lithotrophicus ES-1 (ES-1) grows on FeCO3 or FeS at oxic-anoxic interfaces at circumneutral pH, and the ES-1-mediated Fe(II) oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1's ability to oxidize Fe(II) remain unknown. Survey of the ES-1 genome for candidate genes for microbial extracellular Fe(II) oxidation revealed that it contained a three-gene cluster encoding homologs of Shewanella oneidensis MR-1 (MR-1) MtrA, MtrB, and CymA that are involved in extracellular Fe(III) reduction. Homologs of MtrA and MtrB were also previously shown to be involved in extracellular Fe(II) oxidation by Rhodopseudomonas palustris TIE-1. To distinguish them from those found in MR-1, the identified homologs were named MtoAB and CymA(ES-1). Cloned mtoA partially complemented an MR-1 mutant without MtrA with regards to ferrihydrite reduction. Characterization of purified MtoA showed that it was a decaheme c-type cytochrome and oxidized soluble Fe(ll). Oxidation of Fe(II) by MtoA was pH- and Fe(II)-complexing ligand-dependent. Under conditions tested, MtoA oxidized Fe(II) from pH 7 to pH 9 with the optimal rate at pH 9. MtoA oxidized Fe(II) complexed with different ligands at different rates. The reaction rates followed the order Fe(II)Cl-2 > Fe(II) citrate > Fe(II) NTA > Fe(II) EDTA with the second-order rate constants ranging from 6.3 x 10(-3) mu M-1 s(-1) for oxidation of Fe(II)Cl-2 to 1.0 x 10(-3) mu M-1 s(-1) for oxidation of Fe(II) EDTA. Thermodynamic modeling showed that redox reaction rates for the different Fe(II)-complexes correlated with their respective estimated reaction-free energies. Collectively, these results demonstrate that MtoA is a functional Fe(II)-oxidizing protein that, by working in concert with MtoB and CymA(ES-1), may oxidize Fe(II) at the bacterial surface and transfer released electrons across the bacterial cell envelope to the quinone pool in the inner membrane during extracellular Fe(II) oxidation by ES-1.