Characterization of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth.

Christopher R Beckwith,Thomas A Clarke,Matthew Lawes,Marcus J Edwards,Julea N Butt,David J Richardson,Liang Shi

doi:10.3389/fmicb.2015.00332

Christopher R Beckwith, Thomas A Clarke + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2015.00332

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Abstract

The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidized at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (PMF) (for ATP synthesis) and NADH for autotrophic processes such as carbon fixation. As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical, and crystallographic characterization of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochrome c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.

Highlights

The potential for bacteria to utilize iron as an energy source has been widely recognized in recent years (Bird et al, 2011; Konhauser et al, 2011)
Edman degradation (PNAC facility, Cambridge UK) revealed the Nterminal sequence began AVDVD, matching the cleavage site predicted by SignalP (Petersen et al, 2011) and pyridine hemochrome assays revealed that the sample of MtoD contained approximately stoichiometric ratio of 0.85 heme: protein, giving an ε410 coefficient of 105.2 mM−1 cm−1
The energy source for both NADH and ATP production is generated from the liberation of electrons obtained from the oxidation of iron at the cell surface

Summary

Introduction

The potential for bacteria to utilize iron as an energy source has been widely recognized in recent years (Bird et al, 2011; Konhauser et al, 2011). The role of MtoD in iron oxidation (Rawlings et al, 1999; Ferguson and Ingledew, 2008; Mishra and Rhee, 2014); the marine stalk-forming Gallionella ferruginea and the freshwater Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1 (Hallbeck et al, 1993; Emerson and Moyer, 1997) The genomes of these organisms have been sequenced revealing a range of putative metabolic pathways (Emerson et al, 2013) and analysis of these pathways poses a range of interesting questions: how do the bacteria extract the electrons from the ferrous iron, and how are those electrons coupled to the generation of NADH and a proton motive force (PMF)?. These diheme cytochromes transfer the electrons down divergent routes, either to a cytochrome bc complex where electrons enter the quinol pool to generate NADH, or an aa oxidase where oxygen is reduced to water together with the transport of protons across the membrane (Bonnefoy and Holmes, 2012; Roger et al, 2012)

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