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• W3015560610 abstract "•The 20 hemes of a 3-component complex are arranged to move electrons across 185 Å•A β-barrel and 10-heme cytochrome form an insulated transmembrane nanowire•An extracellular 10-heme cytochrome has a large surface area for electron exchange•The hemes of both cytochromes are packed with a maximum inter-heme distance of 8 Å A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes. A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes. The outer membranes of Gram-negative bacteria are naturally insulative and prevent the indiscriminate exchange of electrons between the cell and environment. However, a number of important bacterial processes require the conductance of electrons across the outer membrane. These include the transfer of intracellularly derived electrons out of the cell to external electron acceptors or the import of electrons into the cell from extracellular electron donors in order to support the formation of reducing equivalents for carbon fixation (Shi et al., 2016Shi L. Dong H. Reguera G. Beyenal H. Lu A. Liu J. Yu H.Q. Fredrickson J.K. Extracellular electron transfer mechanisms between microorganisms and minerals.Nat. Rev. Microbiol. 2016; 14: 651-662Crossref PubMed Scopus (868) Google Scholar). The ability to directly transfer electrons into and out of bacterial cells is also of increasing interest for biotechnological applications including microbial fuel cells, microbial electrosynthesis, unbalanced fermentation, and bio-electronic interfaces (Bursac et al., 2017Bursac T. Gralnick J.A. Gescher J. Acetoin production via unbalanced fermentation in Shewanella oneidensis.Biotechnol. Bioeng. 2017; 114: 1283-1289Crossref PubMed Scopus (52) Google Scholar, Lovley, 2012Lovley D.R. Electromicrobiology.Annu. Rev. Microbiol. 2012; 66: 391-409Crossref PubMed Scopus (531) Google Scholar, Rabaey and Rozendal, 2010Rabaey K. Rozendal R.A. Microbial electrosynthesis - revisiting the electrical route for microbial production.Nat. Rev. Microbiol. 2010; 8: 706-716Crossref PubMed Scopus (1115) Google Scholar). Mechanistically, this process requires electrons to be transported across the outer membrane of the cell in a controlled pathway that prevents detrimental redox side reactions, such as the generation of reactive oxygen species. Thus, an “insulated” electron transfer complex is required. The Gram-negative Shewanella is one of the most studied bacterial genera capable of transferring electrons out of the cell to solid-phase Fe(III) and Mn(IV) minerals in order to support anaerobic respiration (Beblawy et al., 2018Beblawy S. Bursac T. Paquete C. Louro R. Clarke T.A. Gescher J. Extracellular reduction of solid electron acceptors by Shewanella oneidensis.Mol. Microbiol. 2018; 109: 571-583Crossref PubMed Scopus (60) Google Scholar, Edwards et al., 2018Edwards M.J. White G.F. Lockwood C.W. Lawes M.C. Martel A. Harris G. Scott D.J. Richardson D.J. Butt J.N. Clarke T.A. Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.J. Biol. Chem. 2018; 293: 8103-8112Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We have previously purified an icosaheme protein complex comprising three subunits (MtrA, MtrB, MtrC) from the outer membrane of Shewanella oneidensis and shown that this Mtr complex is able to conduct electrons bidirectionally across proteoliposome bilayers sustaining electron transport rates over 8,500 e s–1 (White et al., 2013White G.F. Shi Z. Shi L. Wang Z. Dohnalkova A.C. Marshall M.J. Fredrickson J.K. Zachara J.M. Butt J.N. Richardson D.J. Clarke T.A. Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals.Proc. Natl. Acad. Sci. USA. 2013; 110: 6346-6351Crossref PubMed Scopus (147) Google Scholar). In liposome studies, the direction of electron transfer is dependent on the relative redox potentials across the membrane. Extraliposomal sodium dithionite is capable of reducing intraliposomal methyl viologen or small tetraheme cytochrome (STC), which can then be used to reduce extravesicular iron oxides and chelates (Edwards et al., 2018Edwards M.J. White G.F. Lockwood C.W. Lawes M.C. Martel A. Harris G. Scott D.J. Richardson D.J. Butt J.N. Clarke T.A. Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.J. Biol. Chem. 2018; 293: 8103-8112Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, White et al., 2013White G.F. Shi Z. Shi L. Wang Z. Dohnalkova A.C. Marshall M.J. Fredrickson J.K. Zachara J.M. Butt J.N. Richardson D.J. Clarke T.A. Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals.Proc. Natl. Acad. Sci. USA. 2013; 110: 6346-6351Crossref PubMed Scopus (147) Google Scholar). The reduction potentials of the 20 hemes in the S. oneidensis Mtr complex span from approximately 0 to −400 mV versus SHE. This allows the complex to accept electrons from the menaquinol dehydrogenase CymA, via the periplasmic cytochromes small tetraheme cytochrome (STC) and periplasmic fumarate reductase (FccA), and transfer them to extracellular soluble and insoluble electron acceptors (Figure S1A). Within this complex, the ten heme MtrC is the cell-surface module required when solid-phase minerals serve as terminal electron acceptors (Coursolle and Gralnick, 2010Coursolle D. Gralnick J.A. Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1.Mol. Microbiol. 2010; 77: 995-1008PubMed Google Scholar, Hartshorne et al., 2007Hartshorne R.S. Jepson B.N. Clarke T.A. Field S.J. Fredrickson J. Zachara J. Shi L. Butt J.N. Richardson D.J. Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors.J. Biol. Inorg. Chem. 2007; 12: 1083-1094Crossref PubMed Scopus (174) Google Scholar). MtrAB is proposed to span the outer membrane and provide electrical connection between the catabolic electron transfer network within the cell, and the electron dispersing MtrC outside the cell (Edwards et al., 2018Edwards M.J. White G.F. Lockwood C.W. Lawes M.C. Martel A. Harris G. Scott D.J. Richardson D.J. Butt J.N. Clarke T.A. Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.J. Biol. Chem. 2018; 293: 8103-8112Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Despite these findings, critical information about the complex, including the topology and interactions of all three components, has been missing until now. Here, we present the structure of an Mtr complex at 2.7 Å resolution. This structure reveals MtrAB to be a naturally insulated molecular wire that is able to conduct electrons across the lipidic outer membrane and deliver them to MtrC, which positions its hemes to optimize electron distribution to extracellular redox partners. Despite multiple attempts, the Mtr complex from S. oneidensis could not be crystallized. However, homologous Mtr complexes are produced endogenously by different Shewanella species (Fredrickson et al., 2008Fredrickson J.K. Romine M.F. Beliaev A.S. Auchtung J.M. Driscoll M.E. Gardner T.S. Nealson K.H. Osterman A.L. Pinchuk G. Reed J.L. et al.Towards environmental systems biology of Shewanella.Nat. Rev. Microbiol. 2008; 6: 592-603Crossref PubMed Scopus (722) Google Scholar) and screening of several of these provided a single crystallizable hit. The Mtr complex from Shewanella baltica OS185 produced crystals that diffracted to a final resolution of 2.7 Å, and data were phased utilizing the anomalous signal from the 20 iron atoms of the c-type hemes (Table 1; Figure S1). The crystal structure reveals a heterotrimeric complex that consists of two cytochromes, MtrC and MtrA, and a beta-barrel protein, MtrB, that sheathes and insulates MtrA (Figure 1A). A network of 20 bis-His coordinated hemes spans the complex, forming an electron transfer pathway of 185 Å (Figure 1B). The hemes of MtrA and MtrC are annotated in order of the CxxCH binding motifs to which they are connected, e.g., heme A1 binds to the first motif in the MtrA amino acid sequence and heme C1 binds to the first motif in the MtrC sequence (Figures 1B and 2A ).Table 1Data Collection and Refinement StatisticsData CollectionMtrC SADMtrCAB SADMtrCAB NativeaStatistics for data collection prior to anisotropic correction utilizing STARANISO. Data were truncated along the surface defined by I/σ(I) = 1.2. Corrected data were used for subsequent refinement.Space groupP 21 21 2C 2 2 21C 2 2 21Cell dimensionsa, b, c (Å)90.52, 291.50, 87.20209.90, 235.16, 98.42212.04, 234.17, 99.19α, β, γ (°) (°)90.00, 90.00, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.00Resolution (Å)90.52–2.29 (2.35–2.29)117.58–3.41 (3.50–3.41)106.02–2.70 (2.81–2.70)Rmerge0.098 (1.790)0.143 (0.879)0.115 (2.194)CC1/21.0 (0.48)1.0 (0.49)1.00 (0.73)I / σI17.0 (1.2)9.5 (2.2)9.9 (0.8)Completeness (%)99.5 (98.2)99.5 (99.9)99.0 (97.5)Redundancy12.6 (10.2)6.4 (6.4)5.8 (5.4)RefinementResolution (Å)87.20–2.2973.25–2.70No. reflections104,18749,734Rwork / Rfree0.183/0.2280.224/0.257No. atomsProtein13,42422,512Ligand/ion1,3061496Water91422B factorsProtein53.0063.11Ligand/ion46.5054.52Water51.7737.58RMSDsBond lengths (Å)0.0040.013Bond angles (°)0.7801.753Each dataset was collected from a single crystal. Values in parentheses are for highest-resolution shell.a Statistics for data collection prior to anisotropic correction utilizing STARANISO. Data were truncated along the surface defined by I/σ(I) = 1.2. Corrected data were used for subsequent refinement. Open table in a new tab Figure 2Structural Features of MtrAShow full caption(A) Cartoon of MtrA with hemes shown as sticks. Hemes are numbered as in Figure 1.(B) Heme arrangement within MtrA. Minimum electron transfer distances between the porphyrin rings of adjacent hemes are shown.(C) Flexibility of MtrA. MtrB is shown in grey in cartoon representation. MtrA is shown in cartoon representation and is coloured with a gradient based upon calculated B factors, from low (∼30 Å2) shown in blue to high (∼160 Å2) shown in orange. See also Figures S2 and S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Each dataset was collected from a single crystal. Values in parentheses are for highest-resolution shell. (A) Cartoon of MtrA with hemes shown as sticks. Hemes are numbered as in Figure 1. (B) Heme arrangement within MtrA. Minimum electron transfer distances between the porphyrin rings of adjacent hemes are shown. (C) Flexibility of MtrA. MtrB is shown in grey in cartoon representation. MtrA is shown in cartoon representation and is coloured with a gradient based upon calculated B factors, from low (∼30 Å2) shown in blue to high (∼160 Å2) shown in orange. See also Figures S2 and S3. Hemes A1 to A10 of MtrA are arranged such that neighboring pairs have alternating parallel and perpendicular porphyrin ring planes for which the closest edge-edge distances lie between 3.9 and 6.5 Å (Figure 2B). Similar configurations are found in the heme chains of smaller cytochromes from Shewanella and other bacteria. The heme chain of STC (Leys et al., 2002Leys D. Meyer T.E. Tsapin A.S. Nealson K.H. Cusanovich M.A. Van Beeumen J.J. Crystal structures at atomic resolution reveal the novel concept of “electron-harvesting” as a role for the small tetraheme cytochrome c.J. Biol. Chem. 2002; 277: 35703-35711Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) of Shewanella sp. can be superposed over hemes A2–A5 and hemes A6–A9 of MtrA with root-mean-square deviation (RMSD) of 1.52 Å and 1.64 Å, respectively. The pentaheme chain of NrfB from Escherichia coli (Clarke et al., 2007Clarke T.A. Cole J.A. Richardson D.J. Hemmings A.M. The crystal structure of the pentahaem c-type cytochrome NrfB and characterization of its solution-state interaction with the pentahaem nitrite reductase NrfA.Biochem. J. 2007; 406: 19-30Crossref PubMed Scopus (61) Google Scholar) can be superposed over hemes A1–A5 of MtrA with an RMSD of 1.74 Å (Clarke et al., 2007Clarke T.A. Cole J.A. Richardson D.J. Hemmings A.M. The crystal structure of the pentahaem c-type cytochrome NrfB and characterization of its solution-state interaction with the pentahaem nitrite reductase NrfA.Biochem. J. 2007; 406: 19-30Crossref PubMed Scopus (61) Google Scholar, Leys et al., 2002Leys D. Meyer T.E. Tsapin A.S. Nealson K.H. Cusanovich M.A. Van Beeumen J.J. Crystal structures at atomic resolution reveal the novel concept of “electron-harvesting” as a role for the small tetraheme cytochrome c.J. Biol. Chem. 2002; 277: 35703-35711Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) (Figure S2A). The sequence identity between STC and MtrA in the aligned regions is only 22%–23%, and the corresponding sequence identity between NrfB and MtrA is only 33%. However, the conservation of the heme arrangement observed in these structures suggests that STC, NrfB, and MtrA might share a common ancestor, with MtrA arising from a gene duplication. The arrangement of MtrA heme A9 and A10 cannot be superposed on hemes from either STC or NrfB, possibly because the orientation of heme A10 has altered to facilitate electron transfer to extracellular MtrC. The hemes of STC and NrfB are redox active within the same potential window as MtrA, specifically 0 to −400 mV versus SHE (Clarke et al., 2004Clarke T.A. Dennison V. Seward H.E. Burlat B. Cole J.A. Hemmings A.M. Richardson D.J. Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex.J. Biol. Chem. 2004; 279: 41333-41339Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, Firer-Sherwood et al., 2008Firer-Sherwood M. Pulcu G.S. Elliott S.J. Electrochemical interrogations of the Mtr cytochromes from Shewanella: opening a potential window.J. Biol. Inorg. Chem. 2008; 13: 849-854Crossref PubMed Scopus (135) Google Scholar), and a maximum electron flux of 3 × 106 s–1 though hemes 1–4 of STC has been calculated (Jiang et al., 2017Jiang X. Futera Z. Ali M.E. Gajdos F. von Rudorff G.F. Carof A. Breuer M. Blumberger J. Cysteine Linkages Accelerate Electron Flow through Tetra-Heme Protein STC.J. Am. Chem. Soc. 2017; 139: 17237-17240Crossref PubMed Scopus (29) Google Scholar) in agreement with measurements of heme-heme electron transfer rates in that protein (van Wonderen, 2019van Wonderen Jessica Ultrafast Light-Driven Electron Transfer in a Ru(II)tris(bipyridine)-Labeled Multiheme Cytochrome.Journal American Chemical Society. 2019; : 15190https://doi.org/10.1021/jacs.9b06858Crossref PubMed Scopus (21) Google Scholar). Thus, MtrA may support similarly rapid electron transfer across the 80 Å heme chain between heme A1 and heme A10. This distance is more than sufficient to facilitate the collection of electrons originating within the periplasm, transport across the ∼40 Å outer cell membrane and delivery to the extracellular environment. The MtrA polypeptide has very little secondary structure, with only 20% of the polypeptide chain composed of helices and the remaining 80% consisting of flexible loops (Figure 2A; Figure S2). This is consistent with previous small-angle X-ray scattering (SAXS) analysis of isolated MtrA in solution (Firer-Sherwood et al., 2011Firer-Sherwood M.A. Ando N. Drennan C.L. Elliott S.J. Solution-based structural analysis of the decaheme cytochrome, MtrA, by small-angle X-ray scattering and analytical ultracentrifugation.J. Phys. Chem. B. 2011; 115: 11208-11214Crossref PubMed Scopus (30) Google Scholar). Kratky analysis of these data revealed a peak at low scattering angles, consistent with a folded protein, but increased at higher scattering angles consistent with a flexible MtrA. As a component of the Mtr crystal structure, the flexibility of MtrA can be observed through the temperature (B) factors of the peptide backbone, where higher values are associated with increased chain mobility. The B factors of MtrA increase from the externally facing C terminus to the periplasmic facing N terminus (Figure 2C; Figure S3). Loops in the C-terminal half of the MtrA form hydrogen bonds with internally facing charged MtrB side chains, restricting mobility. In contrast, the interactions between the N-terminal half of MtrA and MtrB are much less extensive, which increases the mobility of the periplasmic facing side of MtrA. The MtrA N terminus is the most mobile region and projects out of MtrB into the periplasmic compartment, with heme A1 located approximately 20 Å inside the periplasm. This could facilitate interactions between heme A1 and soluble periplasmic proteins such as STC and fumarate reductase FccA, which have been previously shown to be electron donors to MtrA (Edwards et al., 2018Edwards M.J. White G.F. Lockwood C.W. Lawes M.C. Martel A. Harris G. Scott D.J. Richardson D.J. Butt J.N. Clarke T.A. Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.J. Biol. Chem. 2018; 293: 8103-8112Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, Sturm et al., 2015Sturm G. Richter K. Doetsch A. Heide H. Louro R.O. Gescher J. A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime.ISME J. 2015; 9: 1802-1811Crossref PubMed Scopus (89) Google Scholar). Solubility of the Mtr complex in the lipidic outer membrane is conferred by MtrB, a hydrophobic barrel comprised of 26 antiparallel β strands (Figures 1A and 3). The interactions between MtrB and MtrA allow MtrA to be positioned across the outer membrane while insulating the MtrA hemes from the outer membrane environment, preventing non-specific reduction of membrane soluble exogenous molecules such as oxygen, which could result in the generation of reactive oxygen species that in turn lead to cellular damage (e.g., lipid peroxidation). The MtrB porin orients MtrA so the heme chain is perpendicular to the membrane and electron transfer away from the cell is optimized. MtrB, with overall dimensions of approximately 70 × 55 × 45 Å, consists of tight turns on the periplasmic face and surface loops extending ∼45 Å on the extracellular side of the membrane. The overall structure of MtrB is similar to that of other outer membrane secretion proteins (e.g., Figure 3). For example, MtrB is approximately the same size as the 26-strand lipopolysaccharide transporter LptD, and the 24-strand pilin subunit transporter FimD (Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens.Structure. 2016; 24: 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, Phan et al., 2011Phan G. Remaut H. Wang T. Allen W.J. Pirker K.F. Lebedev A. Henderson N.S. Geibel S. Volkan E. Yan J. et al.Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.Nature. 2011; 474: 49-53Crossref PubMed Scopus (154) Google Scholar) (Figure 3). The amino acid composition of the extracellular loops gives the MtrB surface a uniform negative charge. Structural modeling of the Shewanella oneidensis MR-1 porins MtrE and DmsF reveals that, while the negatively charged extracellular surfaces are conserved in the MtrB protein family (Figure 4), they are not observed in electrostatic surface maps of the aforementioned FimD and LptD β-barrel proteins (Figure 3). Like MtrB, DmsF and MtrE are components of outer membrane electron transport complexes that contain extracellular catalytic domains, in these cases DmsAB and MtrF, respectively (White et al., 2016White G.F. Edwards M.J. Gomez-Perez L. Richardson D.J. Butt J.N. Clarke T.A. Mechanisms of Bacterial Extracellular Electron Exchange.Adv. Microb. Physiol. 2016; 68: 87-138Crossref PubMed Scopus (104) Google Scholar). It is therefore likely that for MtrB and its homologs these negatively charged residues help in the docking of the extracellular catalytic domains, possibly by preventing the negatively charged Shewanella lipopolysaccharide (Korenevsky et al., 2002Korenevsky A.A. Vinogradov E. Gorby Y. Beveridge T.J. Characterization of the lipopolysaccharides and capsules of Shewanella spp.Appl. Environ. Microbiol. 2002; 68: 4653-4657Crossref PubMed Scopus (59) Google Scholar) from binding to the external surface of the membrane embedded porin:cytochrome complexes. The aperture of the pore defined by MtrB is wider at the periplasmic face (∼30 Å) than at the cell exterior (∼15 Å) where access to the outside is restricted by the folded surface loops (Figures 3 and 5A ). The pore is of sufficient size to allow folded MtrA (∼80 × 30 × 35 Å) to insert into the periplasmic opening of MtrB but prevents MtrA from escaping to the cell exterior, effectively trapping it inside MtrB. Thus, assembly of the full Mtr complex is dependent on “stalled” excretion of MtrA by MtrB, and association with MtrC that is translocated across the outer membrane by the type 2 secretion system (Shi et al., 2008Shi L. Deng S. Marshall M.J. Wang Z. Kennedy D.W. Dohnalkova A.C. Mottaz H.M. Hill E.A. Gorby Y.A. Beliaev A.S. et al.Direct involvement of type II secretion system in extracellular translocation of Shewanella oneidensis outer membrane cytochromes MtrC and OmcA.J. Bacteriol. 2008; 190: 5512-5516Crossref PubMed Scopus (103) Google Scholar). At the N terminus of MtrB, the first four β strands are shorter than the following 22 strands. Predicted to lie within the lipid bilayer, these shorter strands do not interact with MtrA and define a small solvent channel that runs between MtrA and MtrB (Figure 5B). The diameter of the periplasmic facing side of the channel is ∼5 Å, sufficient to allow free diffusion of water in and out of the periplasmic facing side of MtrB. The channel is capped at the extracellular surface of the outer membrane by a small α helix formed by the MtrB surface loop between β strands 1 and 2 (Figure 5C). This helix is stabilized by several hydrogen bonds, including two between Asn71 and the backbone of Tyr133 on β strand 5. The conserved Trp204 of MtrA is positioned underneath the cap and causes a bottleneck that restricts diffusion of charged and polar molecules from the extracellular face of the channel. However, there are polar residues, Asn71 of MtrB, and Gln206 of MtrA, that could stabilize water molecules on either side of the channel and a charged residue Asp107 that could participate in proton exchange. The role of this channel is unclear, but it may allow for proton transport that has been suggested to occur through the Mtr complex during anaerobic respiration (Okamoto et al., 2017Okamoto A. Tokunou Y. Kalathil S. Hashimoto K. Proton Transport in the Outer-Membrane Flavocytochrome Complex Limits the Rate of Extracellular Electron Transport.Angew. Chem. Int. Engl. 2017; 56: 9082-9086Crossref PubMed Scopus (36) Google Scholar). At the cell surface, the surface loops of MtrB largely cover MtrA so that only MtrA residues 284 to 306 and heme A10 are presented for interaction with MtrC (Figure 5D). Interprotein electron transfer from heme A10 is facilitated by the positioning of MtrC heme C5 within an edge-edge distance of 8 Å. The amino acid sequence around heme C5 is highly conserved within the MtrC clade of outer membrane cytochromes (Figure S4). This conserved sequence contains residues that form hydrogen bonds with MtrAB, allowing association of MtrC to the surface of MtrAB. In the absence of MtrC, the exposed edge of the heme A10 porphyrin indicates that reduction of extracellular substrates by MtrAB should be possible and is consistent with previous studies that showed MtrC knockout mutants of S. oneidensis were capable of reduction of soluble Fe(III) chelates but not of insoluble iron oxides (Coursolle and Gralnick, 2010Coursolle D. Gralnick J.A. Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1.Mol. Microbiol. 2010; 77: 995-1008PubMed Google Scholar). To aid initial model building for the Mtr complex, an X-ray crystal structure was obtained for a soluble form of S. baltica OS185 MtrC (MtrCsol). The structure of the monomeric MtrCsol was similar to that of MtrC from S. oneidensis MR-1 described previously (Edwards et al., 2015Edwards M.J. White G.F. Norman M. Tome-Fernandez A. Ainsworth E. Shi L. Fredrickson J.K. Zachara J.M. Butt J.N. Richardson D.J. et al.Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.Sci. Rep. 2015; (Published online July 1, 2015)https://doi.org/10.1038/srep11677Crossref Scopus (111) Google Scholar). MtrCsol consists of 4 domains: two split beta-barrel domains, domains I and III, and two α-helical domains, domains II and IV. These domains serve as a scaffold for 10 bis-His coordinated hemes arranged in a “staggered cross” formation (Edwards et al., 2015Edwards M.J. White G.F. Norman M. Tome-Fernandez A. Ainsworth E. Shi L. Fredrickson J.K. Zachara J.M. Butt J.N. Richardson D.J. et al.Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.Sci. Rep. 2015; (Published online July 1, 2015)https://doi.org/10.1038/srep11677Crossref Scopus (111) Google Scholar). The three MtrCsol monomers within the asymmetric unit of the crystal displayed domain I/II movements relative to domains III/IV. These domain movements centered round a hinge region formed by residues 289–300 located within the alpha helix linking domains II and III. The location of the hinge point suggests this range of motion would not be restricted in the Mtr complex, giving conformational flexibility to MtrC. Analysis by DynDom (Hayward and Lee, 2002Hayward S. Lee R.A. Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50.J. Mol. Graph. Model. 2002; 21: 181-183Crossref PubMed Scopus (250) Google Scholar) showed a maximal rotation of 15 degrees (Figure S5A) and that changes in relative orientation of hemes C1 and C6 were accompanied by <1 Å change in the edge-to-edge distance of the corresponding porphyrin rings (Figure S5B). In the Mtr complex, MtrC is angled on the surface of MtrAB so that the more insulated side of MtrC faces the membrane surface, while the negatively charged heme propionates face toward the environment thereby providing a suitable surface for direct electron transfer to large extracellular substrates (Figure 1). Heme C10 is presented to the environment ∼90 Å above the hydrophobic bilayer core and is located close to the PTPTD amino acid sequence previously identified as a possible hematite binding hydroxylated motif (Lower et al., 2008Lower B.H. Lins R.D. Oestreicher Z. Straatsma T.P. Hochella Jr., M.F. Shi L. Lower S.K. In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype.Environ. Sci. Technol. 2008; 42: 3821-3827Crossref PubMed Scopus (74) Google Scholar); therefore, this may be a primary route for direct electron transfer to insoluble substrates. Previously, modeling of the electronic micro-environments of MtrC suggested that the net driving force between hemes C5 and C10 is rather small, allowing for bi-directional electron transfer through the heme chain formed by C5, C4, C3, C1, C6, C8, C9, and C10 (Figure 1B) (Barrozo et al., 2018Barrozo A. El-Naggar M.Y. Krylov A.I. Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes.Angew. Chem. Int. Engl. 2018; 57: 6805-6809Crossref PubMed Scopus (19) Google Scholar). However, the redox potentials of hemes C2 and C7 are higher than the other hemes, raising the possibility that these hemes serve as bot" @default.
• W3015560610 created "2020-04-17" @default.
• W3015560610 creator A5033611457 @default.
• W3015560610 creator A5070106555 @default.
• W3015560610 creator A5084863408 @default.
• W3015560610 creator A5084979526 @default.
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• W3015560610 date "2020-04-01" @default.
• W3015560610 modified "2023-10-16" @default.
• W3015560610 title "The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire" @default.
• W3015560610 cites W1506664007 @default.
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