Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation

Lockwood, Colin W. J.; Wonderen, Jessica H. van; Edwards, Marcus J.; Piper, Samuel E. H.; White, Gaye F.; Newton‐Payne, Simone; Richardson, David J.; Clarke, Thomas A.; Butt, Julea N.

doi:10.1016/bs.mie.2018.10.011

Cited by 8 publications

(7 citation statements)

References 29 publications

(23 reference statements)

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“…MtrAB when purified and resuspended in detergent micelles was previously (Lockwood et al, 2018) shown to bind to separately purified soluble forms of MtrC but not OmcA, which is an extracellular decaheme cytochrome of S. oneidensis homologous to MtrC. This selective binding led us to anticipate that the MtrC:MtrAB complex would have a structure similar to that of the MTR complex.…”

Section: Resultsmentioning

confidence: 99%

“…Y657C MtrC, lacking the lipid attachment site of wild-type MtrC and carrying a C-terminal Strep II affinity tag, and MtrAB were purified as previously described (Edwards et al, 2018;Lockwood et al, 2018;van Wonderen et al, under review) after overexpression from pBAD202D/TOPO vectors carried by a strain of Shewanella oneidensis MR-1 lacking the mtr operon. Cys-directed labeling of Y657C MtrC with [Ru(4bromomethyl-4 -methylbipyridine)(2,2 -bipyridine) 2 ](PF 6 ) 2 (HetCat, Switzerland) was performed as described previously (van Wonderen et al, 2018, under review).…”

Section: Protein Purification and Biochemical Analysesmentioning

confidence: 99%

“…Previously, we reported (Lockwood et al, 2018) that in vitro mixing of purified MtrAB and soluble forms of MtrC results in the spontaneous formation of a stable high-affinity complex. Formation of the complex was evidenced by native PAGE and analytical ultracentrifugation with both techniques describing a complex of approximately 210 kDa that is indicative of a 1:1 association of soluble MtrC (approximately 80 kDa) and MtrAB (approximately 120 kDa).…”

Section: Introductionmentioning

confidence: 99%

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Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

et al. 2021

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Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.

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Section: Resultsmentioning

confidence: 99%

Section: Protein Purification and Biochemical Analysesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

et al. 2021

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“…Protein Preparation and Characterization. Ru-MtrC Met8, Ru-MtrC His8, and H561M MtrC were prepared using previously explained methods (53,54) and described in detail in SI Appendix. Potentiometric titration as described in ref.…”

Section: Methodsmentioning

confidence: 99%

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme

Wonderen

Adamczyk

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme–heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis. We observed rates of heme-to-heme electron transfer on the order of 109 s−1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser–Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-μs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.

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“…Metal reduction occurs on the external face of the outer membrane by the MtrC, MtrF, or OmcA subunits. The existence of the MtrAB/OmcA complex is still debated, albeit OmcA can functionally replace the MtrC cytochrome (Beblawy et al 2018;Lockwood et al 2018). The OmcA cytochrome is thus represented as interacting with MtrC, the sole in vitro detected interaction (Beblawy et al 2018).…”

Section: Discussionmentioning

confidence: 99%

TheShewanellagenus: ubiquitous organisms sustaining and preserving aquatic ecosystems

Lemaire

Méjean

Iobbi‐Nivol

2020

FEMS Microbiology Reviews

109

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The Gram-negative Shewanella bacterial genus currently includes about 70 species of mostly aquatic γ-proteobacteria, which were isolated around the globe in a multitude of environments such as surface freshwater and the deepest marine trenches. Their survival in such a wide range of ecological niches is due to their impressive physiological and respiratory versatility. Some strains are among the organisms with the highest number of respiratory systems, depending on a complex and rich metabolic network. Implicated in the recycling of organic and inorganic matter, they are important components of organism-rich oxic/anoxic interfaces, but they also belong to the microflora of a broad group of eukaryotes from metazoans to green algae. Examples of long-term biological interactions like mutualism or pathogeny have been described, although molecular determinants of such symbioses are still poorly understood. Some of these bacteria are key organisms for various biotechnological applications, especially the bioremediation of hydrocarbons and metallic pollutants. The natural ability of these prokaryotes to thrive and detoxify deleterious compounds explains their use in wastewater treatment, their use in energy generation by microbial fuel cells and their importance for resilience of aquatic ecosystems.

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Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation

Cited by 8 publications

References 29 publications

Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme

TheShewanellagenus: ubiquitous organisms sustaining and preserving aquatic ecosystems

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