Identification and characterization of M to A: A decaheme c-type cytochrome of the neutrophilic Fe(ll)-oxidizing bacterium Sideroxydans lithotrophicus ES-1

Abstract

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 Fed I) oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1's ability to oxidize Fed I) remain unknown. Survey of the ES-1 genome for candidate genes for microbial extracellular Fed I) 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 Fed 11) reduction. Homologs of MtrA and MtrB were also previously shown to be involved in extracellular Fed I) oxidation by Rhodopseudomonas palustrisJ\E-1. To distinguish them from those found in MR-1, the identified homologs were named MtoAB and CymAEs-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 Fed I). Oxidation of Fed I) by MtoA was pH- and Fe(ll)-complexing ligand-dependent. Under conditions tested, MtoA oxidized Fed I) from pH 7 to pH 9 with the optimal rate at pH 9. MtoA oxidized Fe(ll) complexed with different ligands at different rates. The reaction rates followed the order Fe(ll)CI2 > Fe(ll)-citrate> Fe(ll)-NTA> FedD-EDTA with the second-order rate constants ranging from 6.3×10-3 μM-1 s-1 for oxidation of Fe(ll)Cl2 to 1.0×10-3 μM-1 s-1 for oxidation of FedD-EDTA. Thermodynamic modeling showed that redox reaction rates for the different Fe(ll)-complexes correlated with their respective estimated reaction-free energies. Collectively, these results demonstrate that MtoA is a functional Fe(ll)-oxidizing protein that, by working in concert with MtoB and CymAES-1, may oxidize Fed I) at the bacterial surface and transfer released electrons across the bacterial cell envelope to the quinone pool in the inner membrane during extracellular Fed I) oxidation by ES-1. © 2012 Liu, Wang, Belchik, Edwards, Liu, Kennedy, Merkley, Lipton, Butt, Richardson, Zachara, Fredrickson, Rosso and Shi.