Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.

Marcus J Edwards,David J Richardson,John M Zachara,Julea N Butt,Emma Ainsworth,Gaye F White,Jim K Fredrickson,Liang Shi,Alice Tome-Fernandez,Michael Norman,Thomas A Clarke

doi:10.1038/srep11677

Marcus J Edwards, David J Richardson + Show 9 more

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https://doi.org/10.1038/srep11677

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Abstract

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.

Highlights

Many species of Gram-negative bacteria are capable of coupling anaerobic growth to the extracellular respiration of insoluble minerals containing Fe(III) and Mn(IV) or artificial electrodes
The outer membrane cytochromes MtrC, OmcA and MtrF are exported to the extracellular cell surface by the type II secretion system[11] and S. oneidensis Δ omcA-Δ mtrC double mutants are severely compromised for respiratory mineral Fe(III) reduction and electron transfer to anodes in microbial fuel cells
The MtrC outer membrane multiheme cytochromes (OMMC) clade is spilt into two sub-clades groups; the first MtrC group, MtrC1, contains two CX5–8C motifs while the second, MtrC2, contains just a single CX8C motif in the sequence corresponding to domain III and is the group that contains S. oneidensis MR-1 (Fig. 1)

Summary

Results

The S. oneidensis MtrC CX8C motif is required for aerobic growth. Comparative amino acid sequence analysis of the members of the currently available OMMC clades reveals that all OMMC contain a highly conserved CX8–15C within the sequence corresponding to domain III17. The anaerobic growth profiles of the two S. oneidensis constructs were similar in the presence and absence of arabinose, suggesting the extended lag phase observed under aerobic conditions was due to the loss of the CX8C motif from the membrane bound MtrC in the presence of oxygen. The conserved cysteines 444 and 453 of the CX8C motif form a single disulfide within domain III of MtrC This disulfide is formed between a loop of 8 amino acids that contains a solvent exposed phenylalanine and valine (Fig. 3D). OMMC-FMN interactions were observed after incubation of MtrF or UndA with FMN and reduced glutathione (Fig. 5C,D) , the fluorescence emission intensity at 525 nm was lower than that recorded for MtrC-FMN or OmcA-FMN, and the signal to noise ratio was higher due to background scattering effects

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