Overexpression of Multi-Heme C-type Cytochromes

Shi, Liang; Lin, Jiann-Trzwo; Markillie, Lye Meng; Squier, Thomas C.; Hooker, Brian S.

doi:10.2144/05382pt01

Cited by 55 publications

(72 citation statements)

References 12 publications

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“…Our view is that any change will be negligible since we find MtrC redox activity occurs over a similar potential range when MtrC is in solution, monitored using UV-vis spectropotentiometry, and when it is adsorbed on a solid surface, probed using PFV. It is interesting to note that whole-cell voltammetry of Shewanella species anaerobically grown (conditions where we would expect MtrC to be synthesized) and adsorbed on glassy carbon electrodes revealed reversible reduction and oxidation peaks centered at approximately À100 mV (vs. SHE) [24,25], a similar behavior to that presented here for MtrC. This suggests that intact cells with MtrC exposed on the outer-cell surface exchange electrons with solid substrates over a similar potential window as the redox chemistry determined here for the purified protein.…”

Section: Discussionmentioning

confidence: 99%

“…Cultures of the S. oneidensis MR-1 expression strain LS306, encoding MtrC with a C-terminus tag [21,25], were grown aerobically in Luria-Bertani medium (containing 50 lg mL À1 kanamycin) at 303 K overnight. For scale-up, each initial overnight culture (10 mL) was used to inoculate 1 L of fresh Luria-Bertani medium (containing 50 lg mL À1 kanamycin).…”

Section: Growth Conditions and Protein Purificationmentioning

confidence: 99%

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Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

et al. 2007

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Growth Conditions and Protein Purificationmentioning

confidence: 99%

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

et al. 2007

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“…Cultures were grown aerobically in Luria-Bertani medium (30 μg mL −1 kanamycin), and mtrF expression was induced by the addition of 1 mM arabinose. Purification of MtrF, kinetic, EPR, MCD, and PFV analysis were similar to those described previously for studies on MtrC and OmcA (4,31,36). MtrF crystals were obtained by vapor diffusion using a 1∶1 mixture of 5 mg mL −1 MtrF and a mother liquor of 100 mM N-(2-acetamido)iminodiacetate buffer pH 6.5, 15% ethylene glycol, 1.1 M ðNH 4 Þ 2 HPO 4 .…”

Section: Methodsmentioning

confidence: 99%

Structure of a bacterial cell surface decaheme electron conduit

Clarke

Edwards

Gates

et al. 2011

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

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Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of transouter-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.c-type cytochromes | iron respiration | MtrC | multiheme

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“…Several methods have been developed in Escherichia coli and Shewanella oneidensis strain MR-1 to express multiheme cytochromes, such as the decaheme-containing protein MtrA (18,28). In previous studies, MtrA was expressed in the periplasm as a soluble protein and in association with membrane proteins MtrB and MtrC (11,22,26,28).…”

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confidence: 99%

“…In previous studies, MtrA was expressed in the periplasm as a soluble protein and in association with membrane proteins MtrB and MtrC (11,22,26,28). Previous studies have shown that these cytochromes are part of an electron transport chain that allows the organism to extracellularly respire a number of electron acceptors, including poorly soluble electron acceptors such as iron(III).…”

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confidence: 99%

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

et al. 2012

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c Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme ctype cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ϳ35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.

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Overexpression of Multi-Heme C-type Cytochromes

Cited by 55 publications

References 12 publications

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

Structure of a bacterial cell surface decaheme electron conduit

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

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