Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit (original) (raw)
Related papers
PloS one, 2011
Bioelectrochemical systems rely on microorganisms to link complex oxidation/reduction reactions to electrodes. For example, in Shewanella oneidensis strain MR-1, an electron transfer conduit consisting of cytochromes and structural proteins, known as the Mtr respiratory pathway, catalyzes electron flow from cytoplasmic oxidative reactions to electrodes. Reversing this electron flow to drive microbial reductive metabolism offers a possible route for electrosynthesis of high value fuels and chemicals. We examined electron flow from electrodes into Shewanella to determine the feasibility of this process, the molecular components of reductive electron flow, and what driving forces were required. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at 20.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake, while a mutant lacking the periplasmic fumarate reductase FccA was unable to utilize electrodes for fumarate reduction. Deletion of the gene encoding the outer membrane cytochrome-anchoring protein MtrB eliminated 88% of fumarate reduction. A mutant lacking the periplasmic cytochrome MtrA demonstrated more severe defects. Surprisingly, disruption of menC, which prevents menaquinone biosynthesis, eliminated 85% of electron flux. Deletion of the gene encoding the quinone-linked cytochrome CymA had a similar negative effect, which showed that electrons primarily flowed from outer membrane cytochromes into the quinone pool, and back to periplasmic FccA. Soluble redox mediators only partially restored electron transfer in mutants, suggesting that soluble shuttles could not replace periplasmic protein-protein interactions. This work demonstrates that the Mtr pathway can power reductive reactions, shows this conduit is functionally reversible, and provides new evidence for distinct CymA:MtrA and CymA:FccA respiratory units.
JBIC Journal of Biological Inorganic Chemistry, 2007
MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately À500 mV (vs. the standard hydrogen electrode). Across this potential window the UVvis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled lowspin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.
Microbial nanowires are fascinating biological structures allowing bacteria to transport electrons over micrometers for reduction of extracellular substrates. It was recently established that the nanowires of both Shewanella and Geobacter are made of multi-heme proteins, but while Shewanella employs the 20-heme protein complex MtrCAB, Geobacter uses a redox polymer made of the hexa-heme protein OmcS, begging the question which protein architecture is more efficient in terms of long-range electron transfer. Using a multiscale computational approach we find that OmcS supports electron flows about an order of magnitude higher than MtrCAB due to larger heme-heme electronic couplings and better insulation of hemes from the solvent. We show that heme side chains are an essential structural element in both protein complexes accelerating rate-limiting electron tunnelling steps up to 1000-fold. Our results imply that the alternating stacked/T-shaped heme arrangement present in both protein complexes may be an evolutionarily convergent design principle permitting efficient electron transfer over very long distances.
The role of Shewanella oneidensis MR1 outer surface structures in extracellular electron transfer
Electroanalysis, 2010
The ability of the metal reducer Shewanella oneidensis MR-1 to generate electricity in microbial fuel cells (MFCs) depends on the activity of a predicted type IV prepilin peptidase; PilD. Analysis of an S. oneidensis MR-1 pilD mutant indicated that it was deficient in pili production (Msh and type IV) and type II secretion (T2S). The requirement for T2S in metal reduction has been previously identified, but the role of pili remains largely unexplored. To define the role of type IV or Msh pili in electron transfer, mutants that lack one or both pilus biogenesis systems were generated and analyzed; a mutant that lacked flagella was also constructed and tested. All mutants were able to reduce insoluble Fe(III) and to generate current in MFCs, in contrast to the T2S mutant that is deficient in both processes. Our results show that loss of metal reduction in a PilD mutant is due to a T2S deficiency, and therefore the absence of c cytochromes from the outer surface of MR-1 cells, and not the loss of pili or flagella. Furthermore, MR-1 mutants deficient in type IV pili or flagella generated more current than the wild type, even though extracellular riboflavin levels were similar in all strains. This enhanced current generating ability is in contrast to a mutant that lacks the outer membrane c cytochromes, MtrC and OmcA. This mutant generated significantly less current than the wild type in an MFC and was unable to reduce Fe(III). These results indicated that although nanofilaments and soluble mediators may play a role in electron transfer, surface exposure of outer membrane c cytochromes was the determining factor in extracellular electron transfer in S. oneidensis MR-1.
Brain Research, 2011
Shewanella is frequently used as a model microorganism for microbial bioelectrochemical systems. In this study, we used cyclic voltammetry (CV) to investigate extracellular electron transfer mechanisms from S. oneidensis MR-1 (WT) and five deletion mutants: membrane bound cytochrome (∆mtrC/ΔomcA), transmembrane pili (ΔpilM-Q, ΔmshH-Q, and ΔpilM-Q/ΔmshH-Q) and flagella (∆flg). We demonstrate that the formal potentials of mediated and direct electron transfer sites of the derived biofilms can be gained from CVs of the respective biofilms recorded at bioelectrocatlytic (i.e. turnover) and lactate depleted (i.e. non-turnover) conditions. As the biofilms possess only a limited bioelectrocatalytic activity, an advanced data processing procedure, using the open-source software SOAS, was applied. The obtained results indicate that S. oneidensis mutants used in this study are able to bypass hindered direct electron transfer by alternative redox proteins as well as self-mediated pathways.How does Shewanella transfer its electrons to solid acceptors? Using cyclic voltammetry direct and mediated electron transfer of S. oneidensis MR-1 and related mutants are investigated. The subsequent analysis, based on an elaborate open source software data -processing, indicates a correlation of the maximum current density (x-axes of the graph) of the respective mutant and its mediated electron-transfer ability (respective CV- peak height on the y-axes).► Direct (DET) and mediated (MET) electron transfer of S. Oneidensis. ► Analysis on exemplary knock-out mutants. ► Combining cyclic voltammetry with advanced data analysis. ► Indications for differences in the share of DET and MET between mutants detected.
Methods in Enzymology, 2018
Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.
Characterization of an electron conduit between bacteria and the extracellular environment
Proceedings of the National Academy of Sciences, 2009
A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella , deca -heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa -heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular elemen...
Proceedings of the National Academy of Sciences of the United States of America, 2014
Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane ve...
Electron tunneling properties of outer-membrane decaheme cytochromes from Shewanella oneidensis
Geochimica et Cosmochimica Acta, 2007
We have characterized the outer-membrane decaheme cytochromes OmcA and MtrC from Shewanella oneidensis MR-1 at the singlemolecule level using scanning tunneling microscopy (STM) and tunneling spectroscopy (TS). These cytochrome proteins are of great interest because they are thought to mediate bacterial electron transfer reactions in anoxic waters that control the reductive dissolution of oxide minerals. In our study, to characterize the electron transfer properties of these proteins on a model surface, the purified cytochromes were chemically immobilized as molecular monolayers on Au(111) substrates via a recombinant tetra-cysteine sequence as verified by X-ray photoelectron spectroscopy. Atomic force microscopy images confirm the monolayer films were $5-8 nm thick which is consistent with the apparent lateral dimensions of individual cytochrome molecules obtained with STM. Current-voltage TS of single cytochrome molecules revealed that OmcA and MtrC have different abilities to mediate tunneling current despite having otherwise very similar molecular and biochemical properties. These observations suggest that, based on their electron tunneling properties, the two cytochromes could have specific roles during bacterial metal reduction. Additionally, this study establishes single-molecule STM/TS as an effective means for revealing insights into biogeochemical redox processes in the environment.