Proton Transport in the Outer‐Membrane Flavocytochrome Complex Limits the Rate of Extracellular Electron Transport

Okamoto, Akimitsu; Tokunou, Yoshihide; Kalathil, Shafeer; Hashimoto, Kazuhito

doi:10.1002/ange.201704241

Cited by 10 publications

(13 citation statements)

References 35 publications

(20 reference statements)

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“…It is important to note that the model proposed here does not preclude counter-ion flow. Indeed, Okamoto et al (78) recently reported evidence for proton transport associated with EET in the S. oneidensis MtrC and OmcA multiheme cytochromes.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

Subramanian

Pirbadian

El‐Naggar

et al. 2018

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

158

191

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Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.

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Section: Discussionmentioning

confidence: 99%

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

Subramanian

Pirbadian

El‐Naggar

et al. 2018

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

158

191

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“…[15,16] It is also known that electron transfer is usually coupled to proton transfer which causes acidification in the anodic biofilms and in the anolyte with deleterious consequences for the biofilm metabolism and stability. [3,17,18] The fundamental understanding of these processes that involve large proteins (>70 kDa) often with complex redox properties (with ≥10 heme centers in some multihemes cytochromes) is still in its infancy and precludes the optimization of microbial electrochemical technologies. In Gram-negative electroactive bacteria such as Shewanella, several redox proteins have pH-dependent potentials, [19] such as flavocytochrome c 3 (M r = 63.8 kDa) which is one of the most abundant proteins in the periplasm and the only functional fumarate reductase in this microorganism.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical properties of pH-dependent flavocytochrome c3 from Shewanella putrefaciens adsorbed onto unmodified and catechol-modified edge plane pyrolytic graphite electrode

Lebègue

Costa

Fonseca

et al. 2019

Journal of Electroanalytical Chemistry

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properties of pH-dependent flavocytochrome c3 from Shewanella putrefaciens adsorbed onto unmodified and catechol-modified edge plane pyrolytic graphite electrode. Journal of Electroanalytical Chemistry, Elsevier 2019, 847, pp. AbstractThe electroactivity of adsorbed flavocytochrome c 3 , a tetraheme FAD-containing flavoenzyme isolated from the bacterium Shewanella putrefaciens, is investigated by cyclic voltammetry at an edge plane pyrolytic graphite electrode before and after modification with grafted catechol serving as an efficient pH sensor based on a redox readout.Flavocytochrome c 3 adsorption onto the unmodified or modified electrode surface is successfully achieved by cyclic voltammetry (100 consecutive cycles) in a flavocytochrome c 3 solution containing polymyxin as co-adsorbate. The immobilized flavocytochrome c 3 retains its electrochemical activity and its catalytic fumarate reductase activity. The redox activity of the protein arises from its FAD and four hemes cofactors. The experiments evidence that the hemes' redox potential of flavocytochrome c 3 from Shewanella putrefaciens, for which no crystal structure is yet available, depend on pH which is at variance with data from the other strains Shewanella frigidimarina or Shewanella oneidensis.

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“…For confirmation of the rate limitation by the EET process, monitor the effect of adding shuttling electron mediators such as 100 µM anthraquinone-1-sulfonate ( α-AQS). See the section of Representative Results and reference 15…”

Section: Addition Of Deuterium Water To Measure the Kie On The Eet Prmentioning

confidence: 99%

“…The solid line in Figure 4a is the representative result for the microbial current change induced by the D 2 O addition. The addition of 1.0% (v/v) D 2 O sharply decreased the microbial current within 10 s, while almost no current decrease was observed by the addition of H 2 O (dotted line in Figure 4a) 15 . The same tendency was reproduced in at least four separate experiments.…”

mentioning

confidence: 94%

“…In contrast, methods to investigate the impact of EET-associated cation transport, e.g., protons, on EET kinetics in living cells have been scarcely established, despite proton transport across bacterial membranes having a critical role in signaling, homeostasis, and energy production 12,13,14 . In the present study, we describe a technique to examine the impact of proton transport on EET kinetics in the S. oneidensis MR-1 cell using whole-cell electrochemical measurements, which requires the identification of the rate-limiting step in microbial current production 15 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in <em>Shewanella oneidensis</em> MR-1

Tokunou

Hashimoto

Okamoto

2018

JoVE

Self Cite

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Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

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Proton Transport in the Outer‐Membrane Flavocytochrome Complex Limits the Rate of Extracellular Electron Transport

Cited by 10 publications

References 35 publications

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

Electrochemical properties of pH-dependent flavocytochrome c3 from Shewanella putrefaciens adsorbed onto unmodified and catechol-modified edge plane pyrolytic graphite electrode

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in <em>Shewanella oneidensis</em> MR-1

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