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Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1*

  • Daniel Baron
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Edward LaBelle
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Dan Coursolle
    Affiliations
    Department of Biochemistry, Biophysics, and Molecular Biology, University of Minnesota, St. Paul, Minnesota 55108
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  • Jeffrey A. Gralnick
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108

    Department of Microbiology, University of Minnesota, St. Paul, Minnesota 55108
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  • Daniel R. Bond
    Correspondence
    To whom correspondence should be addressed: BioTechnology Institute, University of Minnesota, 140 Gortner, 1479 Gortner Ave, St. Paul, MN 55108. Tel.: 612-624-8619; Fax: 612-625-1700
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108

    Department of Microbiology, University of Minnesota, St. Paul, Minnesota 55108
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant 2T32-GM008347-16 (to D. C.). This work was also supported by Office of Naval Research Grants N000140810166 (to J. A. G.) and N000140810162 (to D. R. B.).
Open AccessPublished:August 06, 2009DOI:https://doi.org/10.1074/jbc.M109.043455
      Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcAmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s−1). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.
      Respiratory electron flow typically occurs at the inner membrane, where oxidation and reduction can be easily linked to intracellular electron carriers and used to generate a membrane potential. However, when the electron acceptor or donor is insoluble, bacteria must possess a mechanism for transferring electrons beyond their inner membrane (
      • Richardson D.J.
      ). This is especially true for Proteobcteria, which have an outer membrane that further insulates cytoplasmic and inner membrane processes from insoluble substrates. Metal oxides (such as Fe(III) and Mn(IV) oxyhydroxides) are well recognized naturally occurring electron acceptors that demand such an electron transfer strategy (
      • Nealson K.H.
      • Saffarini D.
      ,
      • Lovley D.R.
      • Holmes D.E.
      • Nevin K.P.
      ,
      • Gralnick J.A.
      • Newman D.K.
      ).
      Shewanella oneidensis MR-1, a metabolically versatile member of the gammaproteobacteria (
      • Nealson K.H.
      • Scott J.
      The Prokaryotes.
      ), is capable of reducing insoluble metals, and this phenotype has been linked to a collection of interacting multiheme cytochromes spanning the inner membrane, periplasmic space, and outer membrane (
      • Myers J.M.
      • Myers C.R.
      ,
      • Myers J.M.
      • Myers C.R.
      ,
      • Myers J.M.
      • Myers C.R.
      ,
      • Ross D.E.
      • Ruebush S.S.
      • Brantley S.L.
      • Hartshorne R.S.
      • Clarke T.A.
      • Richardson D.J.
      • Tien M.
      ,
      • Shi L.
      • Chen B.
      • Wang Z.
      • Elias D.A.
      • Mayer M.U.
      • Gorby Y.A.
      • Ni S.
      • Lower B.H.
      • Kennedy D.W.
      • Wunschel D.S.
      • Mottaz H.M.
      • Marshall M.J.
      • Hill E.A.
      • Beliaev A.S.
      • Zachara J.M.
      • Fredrickson J.K.
      • Squier T.C.
      ,
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Ruebush S.S.
      • Brantley S.L.
      • Tien M.
      ). There is also evidence that some of these cytochromes decorate the surface of pili-like structures extending from the cell surface (
      • El-Naggar M.Y.
      • Gorby Y.A.
      • Xia W.
      • Nealson K.H.
      ,
      • Gorby Y.A.
      • Yanina S.
      • McLean J.S.
      • Rosso K.M.
      • Moyles D.
      • Dohnalkova A.
      • Beveridge T.J.
      • Chang I.S.
      • Kim B.H.
      • Kim K.S.
      • Culley D.E.
      • Reed S.B.
      • Romine M.F.
      • Saffarini D.A.
      • Hill E.A.
      • Shi L.
      • Elias D.A.
      • Kennedy D.W.
      • Pinchuk G.
      • Watanabe K.
      • Ishii S.
      • Logan B.
      • Nealson K.H.
      • Fredrickson J.K.
      ). Regardless of the ultimate location of the cytochromes, in all models of electron transfer, electrons must hop from these proteins to a solid surface or be transferred to a soluble mediator that can diffuse to a final destination (
      • Newman D.K.
      • Kolter R.
      ,
      • Lies D.P.
      • Hernandez M.E.
      • Kappler A.
      • Mielke R.E.
      • Gralnick J.A.
      • Newman D.K.
      ). Although chelation of a metal oxide is a third option (
      • Haas J.R.
      • DiChristina T.J.
      ,
      • Taillefert M.
      • Beckler J.S.
      • Carey E.
      • Burns J.L.
      • Fennessey C.M.
      • DiChristina T.J.
      ), the fact that Shewanella is able to transfer electrons to solid graphite electrodes (
      • Kim B.H.
      • Ikeda T.
      • Park H.S.
      • Kim H.J.
      • Hyun M.S.
      • Kano K.
      • Takagi K.
      • Tatsumi H.
      ,
      • Kim B.H.
      • Kim H.J.
      • Hyun M.S.
      • Park D.H.
      ,
      • Kim H.J.
      • Hyun M.S.
      • Chang I.S.
      • Kim B.H.
      ,
      • Kim H.J.
      • Park H.S.
      • Hyun M.S.
      • Chang I.S.
      • Kim M.
      • Kim B.H.
      ,
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ) underscores the need for a direct or diffusion-based electron transfer mechanism to link cellular proteins and surfaces.
      Recent work has shown that Shewanella species secrete soluble flavins (FMN and riboflavin) that facilitate electron transfer to both metals and electrodes (
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ,
      • von Canstein H.
      • Ogawa J.
      • Shimizu S.
      • Lloyd J.R.
      ). For example, removal of accumulated soluble flavins decreases the rate of electron transfer by Shewanella biofilms to electrodes over 80%. Consistent with this observation, kinetic measurements with pure MtrC and OmcA (
      • Ross D.E.
      • Brantley S.L.
      • Tien M.
      ) showed that direct reduction of solid metal oxides by these cytochromes was too slow to explain physiological rates of electron transfer, whereas turnover rates of these enzymes with soluble flavins were orders of magnitude larger. These studies suggest that the kinetics of electron transfer from cytochromes on the outer surface of Shewanella to electrodes will be significantly altered in the absence of diffusible mediators (
      • Ross D.E.
      • Ruebush S.S.
      • Brantley S.L.
      • Hartshorne R.S.
      • Clarke T.A.
      • Richardson D.J.
      • Tien M.
      ,
      • Shi L.
      • Chen B.
      • Wang Z.
      • Elias D.A.
      • Mayer M.U.
      • Gorby Y.A.
      • Ni S.
      • Lower B.H.
      • Kennedy D.W.
      • Wunschel D.S.
      • Mottaz H.M.
      • Marshall M.J.
      • Hill E.A.
      • Beliaev A.S.
      • Zachara J.M.
      • Fredrickson J.K.
      • Squier T.C.
      ,
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Zhang H.
      • Tang X.
      • Munske G.R.
      • Zakharova N.
      • Yang L.
      • Zheng C.
      • Wolff M.A.
      • Tolic N.
      • Anderson G.A.
      • Shi L.
      • Marshall M.J.
      • Fredrickson J.K.
      • Bruce J.E.
      ,
      • Wang Z.
      • Liu C.
      • Wang X.
      • Marshall M.J.
      • Zachara J.M.
      • Rosso K.M.
      • Dupuis M.
      • Fredrickson J.K.
      • Heald S.
      • Shi L.
      ,
      • Shi 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.
      • Richardson D.J.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ,
      • Wigginton N.S.
      • Rosso Jr., K.M.M.F.
      ,
      • Shi L.
      • Squier T.C.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Lower B.H.
      • Shi L.
      • Yongsunthon R.
      • Droubay T.C.
      • McCready D.E.
      • Lower S.K.
      ,
      • Kerisit S.
      • Rosso K.M.
      • Dupuis M.
      • Valiev M.
      ,
      • Marshall M.J.
      • Beliaev A.S.
      • Dohnalkova A.C.
      • Kennedy D.W.
      • Shi L.
      • Wang Z.
      • Boyanov M.I.
      • Lai B.
      • Kemner K.M.
      • McLean J.S.
      • Reed S.B.
      • Culley D.E.
      • Bailey V.L.
      • Simonson C.J.
      • Saffarini D.A.
      • Romine M.F.
      • Zachara J.M.
      • Fredrickson J.K.
      ).
      Voltammetry has proven a useful technique for the analysis of electron transfer rates and pathways using purified proteins (
      • Heering H.A.
      • Weiner J.H.
      • Armstrong F.A.
      ,
      • Heering H.A.
      • Hirst J.
      • Armstrong F.A.
      ,
      • Armstrong F.A.
      ,
      • Jeuken L.J.
      • Jones A.K.
      • Chapman S.K.
      • Cecchini G.
      • Armstrong F.A.
      ,
      • Armstrong F.A.
      ) and has recently been extended to the study of intact bacteria (
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ,
      • Srikanth S.
      • Marsili E.
      • Flickinger M.C.
      • Bond D.R.
      ,
      • Marsili E.
      • Rollefson J.B.
      • Baron D.B.
      • Hozalski R.M.
      • Bond D.R.
      ,
      • Richter H.
      • Nevin K.P.
      • Jia H.F.
      • Lowy D.A.
      • Lovley D.R.
      • Tender L.M.
      ). In slow scan rate cyclic voltammetry, the rate of electron transfer from respiring Shewanella biofilms to electrodes rises sharply at the E°′ of riboflavin and FMN (−0.2 V versus SHE)
      The abbreviation used is: SHE
      standard hydrogen electrode.
      2The abbreviation used is: SHE
      standard hydrogen electrode.
      (
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ). Such measurements relating thermodynamic driving force to turnover kinetics would be difficult with whole cell:Fe(III) oxide incubations, which do not allow fine control over the electron acceptor redox potential or real time recording of electron transfer rates. In addition, voltammetry provides tools to observe electron movement under single-turnover conditions (in the absence of electron donor), allowing reversible oxidation and reduction of proteins accessible to the electrode and study of kinetic behavior (
      • Dumas C.
      • Basseguy R.
      • Bergel A.
      ,
      • Fricke K.
      • Harnisch F.
      • Schröder U.
      ).
      In this work, techniques of turnover (sustained electron transfer from cells to electrode in the presence of electron donor) and single turnover (reversible oxidation and reduction in the absence of electron donor) voltammetry were harnessed to investigate the role of outer membrane proteins in electron transfer from Shewanella to electrodes. In all of these studies, intact metabolically active cells were used, along with electrode surfaces known to act as acceptors for Shewanella. The results in the absence of soluble mediators provide evidence that electron transfer between MtrC and OmcA and surfaces requires a higher potential compared with when flavins are present to shuttle electrons to the surface. Mutant analysis also demonstrates that cells possessing different outer membrane cytochromes have differing abilities for direct and mediator-enabled electron transfer.

      DISCUSSION

      Biochemical, genetic, and genomic analyses have shown that Shewanella strains share a conserved suite of cytochromes responsible for transfer of electrons from the inner to the outer membrane, where they are accessible to metals, mediators, or electrodes (
      • Myers J.M.
      • Myers C.R.
      ,
      • Ross D.E.
      • Ruebush S.S.
      • Brantley S.L.
      • Hartshorne R.S.
      • Clarke T.A.
      • Richardson D.J.
      • Tien M.
      ,
      • Ruebush S.S.
      • Brantley S.L.
      • Tien M.
      ,
      • Zhang H.
      • Tang X.
      • Munske G.R.
      • Zakharova N.
      • Yang L.
      • Zheng C.
      • Wolff M.A.
      • Tolic N.
      • Anderson G.A.
      • Shi L.
      • Marshall M.J.
      • Fredrickson J.K.
      • Bruce J.E.
      ,
      • Wang Z.
      • Liu C.
      • Wang X.
      • Marshall M.J.
      • Zachara J.M.
      • Rosso K.M.
      • Dupuis M.
      • Fredrickson J.K.
      • Heald S.
      • Shi L.
      ,
      • Shi 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.
      • Richardson D.J.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ,
      • Shi L.
      • Squier T.C.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Murphy J.N.
      • Saltikov C.W.
      ). Incubations of outer membrane cytochromes isolated from Shewanella have demonstrated that purified versions of these proteins will reduce metals (
      • Ross D.E.
      • Ruebush S.S.
      • Brantley S.L.
      • Hartshorne R.S.
      • Clarke T.A.
      • Richardson D.J.
      • Tien M.
      ,
      • Shi L.
      • Chen B.
      • Wang Z.
      • Elias D.A.
      • Mayer M.U.
      • Gorby Y.A.
      • Ni S.
      • Lower B.H.
      • Kennedy D.W.
      • Wunschel D.S.
      • Mottaz H.M.
      • Marshall M.J.
      • Hill E.A.
      • Beliaev A.S.
      • Zachara J.M.
      • Fredrickson J.K.
      • Squier T.C.
      ,
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Zhang H.
      • Tang X.
      • Munske G.R.
      • Zakharova N.
      • Yang L.
      • Zheng C.
      • Wolff M.A.
      • Tolic N.
      • Anderson G.A.
      • Shi L.
      • Marshall M.J.
      • Fredrickson J.K.
      • Bruce J.E.
      ,
      • Wang Z.
      • Liu C.
      • Wang X.
      • Marshall M.J.
      • Zachara J.M.
      • Rosso K.M.
      • Dupuis M.
      • Fredrickson J.K.
      • Heald S.
      • Shi L.
      ,
      • Shi 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.
      • Richardson D.J.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ,
      • Wigginton N.S.
      • Rosso Jr., K.M.M.F.
      ,
      • Shi L.
      • Squier T.C.
      • Zachara J.M.
      • Fredrickson J.K.
      ,
      • Lower B.H.
      • Shi L.
      • Yongsunthon R.
      • Droubay T.C.
      • McCready D.E.
      • Lower S.K.
      ,
      • Kerisit S.
      • Rosso K.M.
      • Dupuis M.
      • Valiev M.
      ,
      • Marshall M.J.
      • Beliaev A.S.
      • Dohnalkova A.C.
      • Kennedy D.W.
      • Shi L.
      • Wang Z.
      • Boyanov M.I.
      • Lai B.
      • Kemner K.M.
      • McLean J.S.
      • Reed S.B.
      • Culley D.E.
      • Bailey V.L.
      • Simonson C.J.
      • Saffarini D.A.
      • Romine M.F.
      • Zachara J.M.
      • Fredrickson J.K.
      ). The recent finding that Shewanella also secretes soluble flavins that accelerate metal or surface reduction showed that a soluble agent could help solve the problem of the last “hop” from proteins to surfaces (
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ,
      • von Canstein H.
      • Ogawa J.
      • Shimizu S.
      • Lloyd J.R.
      ). In this work, we have provided observations of electron transfer from intact cells without the apparent aid of flavins, shown that this electron transfer occurs at specific potentials, and provided estimates of rate constants for the process in living cells. Thus, both mechanisms of electron transfer may be operational, with the relative contributions determined by flavin concentrations, diffusional distances (for mediated transfer), and driving force (for direct transfer).
      When enzymes (
      • Heering H.A.
      • Weiner J.H.
      • Armstrong F.A.
      ,
      • Heering H.A.
      • Hirst J.
      • Armstrong F.A.
      ,
      • Hirst J.
      • Sucheta A.
      • Ackrell B.A.
      • Armstrong F.A.
      ) or bacteria (
      • Srikanth S.
      • Marsili E.
      • Flickinger M.C.
      • Bond D.R.
      ,
      • Marsili E.
      • Rollefson J.B.
      • Baron D.B.
      • Hozalski R.M.
      • Bond D.R.
      ,
      • Busalmen J.P.
      • Esteve-Nunez A.
      • Feliu J.M.
      ,
      • Busalmen J.P.
      • Esteve-Nunez A.
      • Berna A.
      • Feliu J.M.
      ) are linked to electrodes via pathways with rapid interfacial rates of electron transfer, an increase in applied potential causes an identical increase in the potential of attached enzyme redox center(s). This creates the classic sigmoidal i-V wave, which rises at a single redox potential and plateaus at a limiting current reflecting a limiting enzymatic turnover rate (
      • Richter H.
      • Nevin K.P.
      • Jia H.F.
      • Lowy D.A.
      • Lovley D.R.
      • Tender L.M.
      ). In contrast, the more linear dependence of electron transfer rate with voltage observed in higher potential regions with Shewanella (Figs. 1 and 4) suggested an interaction with slower or mixed interfacial kinetics. The response to soluble flavins in both naturally growing cells and cell films further illustrated that there was an excess of enzyme activity at the electrode but a bottleneck in the electron transfer pathway that could be alleviated by the presence of a soluble redox mediator.
      The results obtained with intact cells of Shewanella allow some comparison with those obtained in vitro with recombinant proteins in two key parameters; apparent midpoint potentials, and estimated electron transfer rates. It is important to note that recombinant proteins are typically purified with affinity tags, expressed without lipid-binding domains, incubated in the presence of detergents, studied with soluble iron ligands, and/or are bound to electrodes with the aid of agents such as polymixin (
      • Shi L.
      • Chen B.
      • Wang Z.
      • Elias D.A.
      • Mayer M.U.
      • Gorby Y.A.
      • Ni S.
      • Lower B.H.
      • Kennedy D.W.
      • Wunschel D.S.
      • Mottaz H.M.
      • Marshall M.J.
      • Hill E.A.
      • Beliaev A.S.
      • Zachara J.M.
      • Fredrickson J.K.
      • Squier T.C.
      ,
      • Wang Z.
      • Liu C.
      • Wang X.
      • Marshall M.J.
      • Zachara J.M.
      • Rosso K.M.
      • Dupuis M.
      • Fredrickson J.K.
      • Heald S.
      • Shi L.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ,
      • Wigginton N.S.
      • Rosso Jr., K.M.M.F.
      ,
      • Eggleston C.M.
      • Voros J.
      • Shi L.
      • Lower B.H.
      • Droubay T.C.
      • Colberg P.J.S.
      ). Such treatments could alter the exposed surface of the protein, increase flexibility, allow closer contact, or facilitate more rapid turnover.
      For example, MtrC has been shown in two separate studies to be reversibly oxidized at basal plane graphite electrodes across a wide potential range, similar to what was observed in our study with whole cells (of over 400 mV). Films of purified MtrC produced broad redox peaks centered at −138 mV (
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ) and −100 mV (
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ). Using cytochromes attached to scanning tunneling microscope tips, an equally wide potential window centered near −200 mV was observed for MtrC (
      • Wigginton N.S.
      • Rosso Jr., K.M.M.F.
      ). Films of OmcA have also been reported to have potentials in the −150 to −100 mV range, using metal oxide or graphite electrodes (
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ,
      • Eggleston C.M.
      • Voros J.
      • Shi L.
      • Lower B.H.
      • Droubay T.C.
      • Colberg P.J.S.
      ).
      In contrast, direct electron transfer from intact Shewanella films to graphite electrodes described here required potentials near 0 V, or at least 100–200 mV more positive than previously reported. Data from Shewanella sp. strain MR-4 biofilms also detected reversible electron transfer reactions in the same 0 V range (
      • Marsili E.
      • Baron D.B.
      • Shikhare I.D.
      • Coursolle D.
      • Gralnick J.A.
      • Bond D.R.
      ). In this work, deletion of specific cytochromes altered the redox behavior of whole cells, which supported the hypothesis that we were observing electron transfer as catalyzed by these well studied outer membrane (or pili-attached) proteins.
      A second comparison is related to rates of electron transfer. Estimates for interfacial rate constants in excess of 100 s−1 have been reported for purified MtrC (
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ). Such rates (in the 100–1000 s−1 range) are highly typical of adsorbed enzymes studied by film voltammetry (
      • Jeuken L.J.
      • Jones A.K.
      • Chapman S.K.
      • Cecchini G.
      • Armstrong F.A.
      ,
      • Baymann F.
      • Barlow N.L.
      • Aubert C.
      • Schoepp-Cothenet B.
      • Leroy G.
      • Armstrong F.A.
      ). Rates for cytochromes interacting with solid electron acceptors are more variable and are often based on measurements during electron transfer to solid substrates (expressed as kcat or rates observed under moderate driving force).
      Using purified OmcA, both Eggleston et al. (
      • Eggleston C.M.
      • Voros J.
      • Shi L.
      • Lower B.H.
      • Droubay T.C.
      • Colberg P.J.S.
      ) and Xiong et al. (
      • Xiong Y.
      • Shi L.
      • Chen B.
      • Mayer M.U.
      • Lower B.H.
      • Londer Y.
      • Bose S.
      • Hochella M.F.
      • Fredrickson J.K.
      • Squier T.C.
      ) observed electron transfer to Fe(III) nanoparticles or oxide-coated electrodes on the order of 10−13 mol e cm−2 s−1, equivalent to ∼0.1 s−1/cytochrome. Incubations with whole Shewanella putrefaciens and Fe(III) nanoparticles produced estimates of 0.001 s−1 (
      • Bonneville S.
      • Behrends T.
      • Van Cappellen P.
      • Hyacinthe C.
      • Roling W.F.M.
      ), although only a percentage of cytochromes were likely reacting with Fe(III), causing this latter rate to represent a lower boundary. Similarly low rates of single-protein turnover to goethite of 0.5–5 s−1 were recently provided by Ross et al. (
      • Ross D.E.
      • Brantley S.L.
      • Tien M.
      ). Because an overpotential of at least 100 mV is present to estimate kcat, ko values for intact cells could be expected to be at least this low.
      Our scan rate analysis data from whole cells provided independent estimates of interfacial electron transfer to external acceptors. Although these values represent averages of all electron transfer reactions involved in direct transfer for an intact Shewanella cell, they also represent a glimpse into the real world conditions experienced by a cell attempting utilize its intact electron transfer system to reduce this surface, with proteins confined to complexes within the membrane. All of our measurements were consistent with rates on the order of 1 s−1 or slower, which were 10–100-fold lower than k0 values observed for pure proteins adsorbed to electrodes (
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Firer-Sherwood M.
      • Pulcu G.S.
      • Elliott S.J.
      ).
      When the maximum number of redox centers able to form a cell-electrode connection and rate constants from our scan rate analysis are combined, a prediction of the exchange current density (j0) for the nonmediated pathway can be made. Assuming that the final step of electron transfer occurs through cytochromes with a size similar to OmcA or MtrC, the most electron transfer sites (regardless of whether cytochromes are on the cell surface or on cell pili) that can possibly be in physical contact with any electrode is on the order of 10−12 mol/cm2 (
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Blanford C.F.
      • Armstrong F.A.
      ). Based on estimated k0 values for this pathway (∼1 s−1), this is equivalent to an exchange current between cells and electrodes of only ∼1 × 10−7 A/cm2.
      Values in this low j0 range require significant overpotentials (of at least ∼100 mV) simply to achieve a forward anodic current of the magnitude attributed to the non-flavin-mediated pathway (∼10 μA/cm2) (
      • Bard A.J.
      • Faulkner L.R.
      ). This requirement for overpotential is compounded by the broad potential window of the cytochromes, meaning that at a potential <0 V, only a fraction of redox sites are experiencing conditions that enable forward electron transfer. These calculations are also consistent with estimates by others (
      • Hartshorne R.S.
      • Jepson B.N.
      • Clarke T.A.
      • Field S.J.
      • Fredrickson J.
      • Zachara J.
      • Shi L.
      • Butt J.N.
      • Richardson D.J.
      ,
      • Xiong Y.
      • Shi L.
      • Chen B.
      • Mayer M.U.
      • Lower B.H.
      • Londer Y.
      • Bose S.
      • Hochella M.F.
      • Fredrickson J.K.
      • Squier T.C.
      ), where films of pure OmcA subjected to moderate driving force supported electron transfer rates equivalent to only ∼1.6 μA/cm2.
      Taken together, the boost in electron transfer in naturally grown biofilms above 0 V (Fig. 1) and the electron transfer observed in the same potential range with adsorbed Shewanella in the absence of flavins (Fig. 4) are likely due to cytochrome-electrode interactions. The relationship between applied potential and electron transfer at higher potentials is consistent with a step in the direct pathway being relatively slow, compared with reactions that supply electrons to this interface. This is also supported by the thin-film behavior relationship, which would be expected if reaction(s) feeding electrons to the interface were faster than the interface reaction itself.
      In contrast, even small amounts of flavins (when present) can facilitate the transfer of electrons to electrodes at lower potentials and cycle back to Shewanella cell surface cytochromes. Diffusion limits the contribution of electron transfer by this mechanism, as shown by the strong relationship between soluble flavin concentration and anodic current (Figs. 1 and 4) and current/protein ratios (Fig. 2).

      Implications for Shewanella

      These results show that redox-active proteins can be present on the outer surface of cells but not interfaced properly with the cell interior (e.g. as seen in MtrC mutants that produce peaks in Fig. 5 but no catalytic activity in Fig. 4). In addition, the cytochromes present on the outer surface appear to experience a bottleneck in electron transfer to solid phase electron acceptors, a conclusion also reached in recent experiments with purified OmcA and MtrC by Ross et al. (
      • Ross D.E.
      • Brantley S.L.
      • Tien M.
      ).
      Molecular computations of multiheme cytochromes (
      • Kerisit S.
      • Rosso K.M.
      • Dupuis M.
      • Valiev M.
      ) have shown that subtle variations in the orientation of hemes relative to a surface can slow rates of electron transfer by the same protein from nearly 100 to 0.01 s−1. Because changes in the form of chelated Fe(III) (
      • Wang Z.
      • Liu C.
      • Wang X.
      • Marshall M.J.
      • Zachara J.M.
      • Rosso K.M.
      • Dupuis M.
      • Fredrickson J.K.
      • Heald S.
      • Shi L.
      ), the nature of oxide surfaces (
      • Eggleston C.M.
      • Voros J.
      • Shi L.
      • Lower B.H.
      • Droubay T.C.
      • Colberg P.J.S.
      ), and even alterations to the orientation of hemes at a surface (
      • Kerisit S.
      • Rosso K.M.
      • Dupuis M.
      • Valiev M.
      ) can dramatically alter electron transfer rates, surfaces other than carbon electrodes may interact differently with the direct transfer pathway of Shewanella.
      In the environment, the diversity of reducible oxide surfaces may select for flavin secretion as a means for cells to gain access to wider range of acceptors, without requiring the organism to express many different cytochromes tuned to a variety of unpredictable surfaces. Thus, flavins could act as a kind of “universal translator,” allowing Shewanella to gain access to a diverse array of redox-active substrates, using only a single cytochrome-based pathway.
      These observations also argue that, under conditions such as microbial fuel cells where anodes stabilize at low (−0.1 V) potentials, Shewanella would not compete with organisms that are capable of higher rates of respiration in the −0.1 V range (
      • Srikanth S.
      • Marsili E.
      • Flickinger M.C.
      • Bond D.R.
      ,
      • Marsili E.
      • Rollefson J.B.
      • Baron D.B.
      • Hozalski R.M.
      • Bond D.R.
      ,
      • Xing D.
      • Zuo Y.
      • Cheng S.
      • Regan J.M.
      • Logan B.E.
      ) unless mediators accumulated to significantly facilitate electron transfer. Alternatively, if an anode was maintained at a higher potential (via use of a lower external resistance or potentiostat control), these observations provide a thermodynamic basis for enrichment of different populations of bacteria, based on the differing responses to potential in this relatively narrow range.

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