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Title: Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective

Abstract

Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. But, the mechanisms associated with EET are complex and involve molecular interactions that take place at the “bionano interface” where biotic/abiotic interactions are usually explored. Our work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties, and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.

Authors:
 [1]; ORCiD logo [2];  [1];  [3];  [4];  [5]
  1. Univ. of New Mexico, Albuquerque, NM (United States). Chemical and Biological Engineering Dept., Center for Micro-engineering Materials
  2. Univ. of New Mexico, Albuquerque, NM (United States). Chemical and Biological Engineering Dept., Center for Micro-engineering Materials; Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  3. J. Craig Venter Inst., San Diego, CA (United States)
  4. J. Craig Venter Inst., San Diego, CA (United States); Univ. of Southern California, Los Angeles, CA (United States). Dept. of Earth Sciences and Biological Sciences
  5. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1411338
Report Number(s):
LA-UR-15-29075
Journal ID: ISSN 1934-8630
Grant/Contract Number:
AC52-06NA25396
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biointerphases
Additional Journal Information:
Journal Volume: 12; Journal Issue: 2; Journal ID: ISSN 1934-8630
Publisher:
American Institute of Physics (AIP)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biological Science; Energy Sciences; Material Science

Citation Formats

Babanova, Sofia, Matanovic, Ivana, Cornejo, Jose, Bretschger, Orianna, Nealson, Kenneth, and Atanassov, Plamen. Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective. United States: N. p., 2017. Web. doi:10.1116/1.4984007.
Babanova, Sofia, Matanovic, Ivana, Cornejo, Jose, Bretschger, Orianna, Nealson, Kenneth, & Atanassov, Plamen. Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective. United States. doi:10.1116/1.4984007.
Babanova, Sofia, Matanovic, Ivana, Cornejo, Jose, Bretschger, Orianna, Nealson, Kenneth, and Atanassov, Plamen. Wed . "Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective". United States. doi:10.1116/1.4984007.
@article{osti_1411338,
title = {Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective},
author = {Babanova, Sofia and Matanovic, Ivana and Cornejo, Jose and Bretschger, Orianna and Nealson, Kenneth and Atanassov, Plamen},
abstractNote = {Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. But, the mechanisms associated with EET are complex and involve molecular interactions that take place at the “bionano interface” where biotic/abiotic interactions are usually explored. Our work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties, and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.},
doi = {10.1116/1.4984007},
journal = {Biointerphases},
number = 2,
volume = 12,
place = {United States},
year = {Wed May 31 00:00:00 EDT 2017},
month = {Wed May 31 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
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  • SUMMARY Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that is capable of using insoluble oxidized metals, such as manganese [Mn(III, IV)] and iron [Fe(III)] oxides and oxyhydroxides, as terminal electron acceptors during anaerobic respiration. The ability of S. oneidensis MR-1 to reduce oxidized Mn and/or Fe has previously been linked to OmcA and MtrC: two decaheme c-type cytochromes that are localized to the outer membrane. To investigate how the electron transport proteins OmcA and MtrC are organized, we expressed and purified recombinant OmcA and MtrC from wild type S. oneidensis MR-1 as well as a mutant that lacked OmcAmore » and MtrC (ΔomcA/mtrC). After purification to the nearly electrophoretic homogeneity from the ΔomcA/mtrC mutant, the recombinant OmcA and MtrC exhibited the characteristics of c-type cytochromes, and each of their polypeptides was confirmed to contain 10 hemes. When purified from wild type cells, endogenous MtrC or OmcA was always co-purified with recombinant OmcA or MtrC, respectively. Fluorescence polarization experiment showed that recombinant OmcA bound to the FlAsH-labeled MtrC with a dissociation constant of 7 ×10-7 M. The purified recombinant OmcA or MtrC alone displayed intrinsic ferric reductase activity with NADH used as an electron donor. Ferric reductase specific activity increased by 35 to 41% when nearly equimolar concentrations of OmcA and MtrC were assayed relative to the two proteins assayed independently. These results demonstrate that OmcA and MtrC directly interact with each other to form a stable complex with high ferric reductase activity.« less
  • In this report, we describe the characterization of two outer-membrane decaheme cytochromes OmcA and MtrC purified from the metal-reducing bacterium Shewanella oneidensis using scanning tunneling microscopy (STM) and tunneling spectroscopy (TS). OmcA and MtrC were solubilized with a common detergent and irreversibly bound to Au (111) substrates as self-assembled cytochrome films. X-ray photoelectron spectroscopy (XPS) verified that OmcA and MtrC were covalently bound to the Au surface via thiol bonds to cysteine residues. Initial STM images show that a layer of detergent covers and protects the cytochrome films. Temporary application of high bias voltage causes the detergent film to reorganizemore » around the tip, opening a window for direct STM imaging of the cytochrome layer underneath. The STM apparent sizes of both OmcA and MtrC are 58 nanometers in diameter consistent with expectations from their molecular masses. Current-voltage TS over individual cytochromes showed that OmcA and MtrC have different abilities to mediate the tunneling current, reflecting differences in their electronic structures. The data suggest that the two cytochromes could have different roles in the electron transport chain during metal reduction.« less
  • Shewanella oneidensis MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration.  A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface.  Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe2O3) thin film, created with oxygen plasma assisted molecular beam epitaxy (MBE), and recombinant MtrC or OmcA molecules coupled to gold substrates.  Force spectra displayed a unique force signature indicative of a specific bond betweenmore » each cytochrome and the hematite surface.  The strength of the OmcA-hematite bond was approximately twice as strong as the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC.  Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite.  The force measurements for the hematite-cytochrome pairs were compared to spectra collected between an iron oxide and S. oneidensis under anaerobic conditions.  There is a strong correlation between the whole cell and pure protein force spectra suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals.  Finally, by comparing the magnitude of binding force for the whole cell vs. pure protein data, we were able to estimate that a single bacterium of S. oneidensis (2 x 0.5 μm) expresses ~104 cytochromes on its outer surface. « less
  • Outer membrane decaheme c-type cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 are extracellular lipoproteins important for dissimilatory reduction of solid metal (hydr)oxides during anaerobic respiration. To investigate the roles of type II secretion system (T2S) in translocation of MtrC and OmcA across outer membrane, we measured the effects of deleting two T2S genes, gspD and gspG, on the secretion of MtrC and OmcA when cells were grown under anaerobic conditions. Deletion of gspD or gspG resulted in slightly yellowish supernatants, different from the pink supernatant of wild type (wt). Comparative proteomic analyses revealed that, although MtrC, OmcA and NrfA,more » a periplasmic nitrite reductase, were present the supernatants of wt and ΔgspD mutant, their peptides counts were much lower in ΔgspD than in wt. Subsequent analyses with heme-staining and Western blot not only confirmed that deletion of gspD or gspG reduced the abundances of MtrC and OmcA in the supernatants, but also revealed that the deletions consequently increased their abundances inside the cells. Complementation of ΔgspG mutant with functional GspG could reverse the effects of deleting gspG on the colors of the supernatants and the abundances of MtrC and OmcA. In contrast, Western results showed that the abundance of NrfA was reduced in the supernatant and the cells of ΔgspD mutant, suggesting that reduced NrfA in the periplasm, where MtrC and OmcA were accumulated, contributed to its reduction in the supernatant. Thus, our results demonstrate at the first time that T2S facilitates translocation of MtrC and OmcA across outer membrane.« less
  • Shewanella Oneidensis MR-1 possesses up to 42 c-type cytochromes with heme content varying between 1 to as many as 37. Among them, the outer-membrane cytochromes, particularly MtrC and OmcA, are suspected to function as terminal reductases and are responsible for its enzymatic catalysis capability. So far, the mechanisms of metal reduction by these outer-membrane cytochromes are unknown. In this work, we report the study of reduction kinetics of a series of Fe(III) complexes with citrate, NTA and EDTA by abiotically reduced MtrC and OmcA using a stopped-flow technique in combination with theoretical computation methods within the framework of the electronmore » transfer theory of Marcus and speciation calculations based on the current thermodynamic database. Stopped-flow kinetic data showed that the reaction was very fast and appeared to proceed in two stages, a fast stage that completes in much less than a second and a slower stage afterwards. For a given complex, the reaction is faster by reduction with MtrC than OmcA, while for a given protein, the reaction completes in the decreasing order of Fe-EDTA > Fe-NTA > Fe-citrate. All the stopped-flow kinetic curves could be modeled by two parallel second-order bimolecular redox reactions with second-order rate constants ranging from 0.872 µM -1s -1 for the fast reaction between MtrC with Fe-EDTA complex to 0.012 µM -1s -1 for the slow reaction between OmcA and Fe-citrate complex. Speciation calculations indicated that at both metal:ligand ratios, 1:1.5 and 1:10, a single dominant ferric complex was responsible for the observed reaction for each ligand and, therefore, the observed dual-reaction pathways was attributed to the differences in the reduction behavior among various heme groups within each protein. The results of redox potential calculations with known thermodynamic data show only small differences on the scale of a few millivolts among the three complexes, suggested that the observed differences in reaction rate cannot be explained by the overall redox reaction free energy. In contrast, reorganization energies calculated based on DFT-COSMO model are substantially different between the complexes, with a larger reorganization energy and therefore a larger activation energy associated with the citrate complex, and progressively smaller ones for the NTA and EDTA complexes. In combination with approximate electronic coupling terms, the theoretical results show good agreement with the observed trend and implicate the reorganization energy as the key factor in the kinetic reaction.« less