Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.681514
Title: Characterisation of cytochrome c MtoD from the iron-oxidising autotroph sideroxydans lithotrophicus ES-1
Author: Beckwith, Chris
ISNI:       0000 0004 5920 696X
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2015
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Abstract:
Despite the discovery of iron-oxidising microorganisms as early as 1836 by Ehrenberg, as described by Harder in 1919, iron-oxidising lithotrophs and their respiratory pathways remain poorly understood. The neutrophilic iron-oxidiser Sideroxydans lithotrophicus ES-1 contains a conserved mtoDAB gene cluster. MtoAB, consisting of a decaheme c-type cytochrome (MtoA) and β-barrel porin (MtoB), is implicated in Fe(II) oxidation at the outer membrane, performing a similar electron transfer function to the well-studied MtrCAB complex from iron-reducing bacterium Shewanella oneidensis, albeit in reverse. MtoD is predicted to be a monoheme class I cytochrome c that is proposed to transfer electrons from MtoA to the predicted inner membrane quinone reductase CymAES-1 (Shi et al. 2012). Very little is understood about the electron transfer pathways in S. lithotrophicus and the current model (Shi et al. 2012) does not propose a mechanism that would allow NADH and ATP to be generated for the fixation of CO2. To probe the potential electron transfer pathways in S. lithotrophicus, MtoD was overexpressed in S. oneidensis, purified and characterised using biophysical, structural and spectroelectrochemical methods. The primary structure of MtoD contains a single CXXCH c-type heme binding motif, but the mature protein sequence lacks methionine, which is the distal ligand to the heme iron in almost all class I cytochromes c. Biophysical characterisation using AUC and SAXS indicated MtoD behaved as a monomer in solution with an approximate molecular weight of 11.5 kDa and the molecular envelope fitted well to previously characterised monoheme cytochromes c. The X-ray crystal structure of MtoD was determined to a resolution of 1.47 Å and revealed a protein fold typical of a class I cytochrome c. Unlike many other class I cytochromes c, MtoD contains a bis-His ligated heme which is solvent exposed on two sides. Redox characterisation of MtoD revealed a midpoint potential of +150 mV vs. SHE for MtoDs c-type heme, which shifted to more negative potentials when adsorbed to SnO2 electrodes, potentially as a result of interaction-induced conformational changes. These redox changes in addition to the atypical structure around the highly exposed heme cofactor make it possible that MtoD could be capable of interacting with multiple redox partners, allowing electron transfer to quinone reductases and cytochrome c oxidase at the inner membrane of S. lithotrophicus. Based on the characterisation of MtoD and an analysis of the c-type cytochromes present in the S. lithotrophicus genome, a new model for electron transfer during aerobic iron lithotrophy is proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.681514  DOI: Not available
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