German Edition: DOI: 10.1002/ange.201800294BioelectronicsInternational Edition: DOI: 10.1002/anie.201800294

Distinct Electron Conductance Regimes in Bacterial DecahemeCytochromesAlexandre Barrozo, Mohamed Y. El-Naggar, and Anna I. Krylov*

Abstract: Shewanella oneidensis MR-1 gains energy byextracellular electron transfer to solid surfaces. They employc-type cytochromes in two Mtr transmembrane complexes,forming a multiheme wire for electron transport across thecellular outer membrane. We investigated electron- and hole-transfer mechanisms in the external terminal of the twocomplexes, MtrC and MtrF. Comparison of computed redoxpotentials with previous voltammetry experiments in distinctenvironments (isolated and electrode-bound conditions ofPFV or in vivo) suggests that these systems function in differentregimes depending on the environment. Analysis of redoxpotential shifts in different regimes indicates strong couplingbetween the hemes via an interplay between direct Coulomband indirect interactions through local structural reorganiza-tion. The latter results in the screening of Coulomb interactionsand explains poor correlation of the strength of the heme-to-heme interactions with the distance between the hemes.

Extracellular electron transfer (EET) is a respiratory strat-egy that allows metal-reducing bacteria, including Shewanellaoneidensis MR-1, to gain energy by coupling the oxidation ofintracellular electron donors to the reduction of electronacceptors outside the cells·[1] This process expands the rangeof suitable oxidants for respiration beyond soluble molecules(for example, O2) that enter the cell to solid-phase mineralssuch as iron and manganese oxides. Along with the global-scale environmental significance of EET, this process is nowbeing exploited in a new generation of renewable energytechnologies incorporating microbial catalysts on electrodesurface for fuel-to-electricity (microbial fuel cells) or elec-tricity-to-fuel (microbial electrosynthesis) conversion.[2, 3]

Multiheme cytochromes are critical for EET in S.oneidensis. The MtrABC porin–cytochrome complex func-tions as a conduit for electrons to cross the cell envelope.

Electrons flow from the periplasmic decaheme cytochromeMtrA to the outer membrane decaheme cytochrome MtrCand the transmembrane porin MtrB facilitates contactbetween the two.[4] If in direct contact with the electronaccepting surface, MtrC and another outer membranedecaheme cytochrome (OmcA) can function as terminalreductases for direct EET to this external surface.[5,6] Thesesurface-exposed cytochromes also contain binding sites forflavins that are known to be secreted by S. oneidensis.[7]

Flavins may function as either cytochrome-bound cofactorsthat enhance EET[8, 9] or as soluble redox shuttles that extendEET to more distant external surfaces.[10] MtrC and OmcAalso localize in micrometer-scale extensions of the outermembrane and periplasm, structures that have been proposedto function as bacterial nanowires for long distance EET.[11,12]

Different mechanisms of EET (direct versus mediated) areassociated with distinct voltammetry signatures observedin vivo.[13] The S. oneidensis genome encodes another Mtrpathway (MtrDEF), which is a paralogue of MtrABC. Thisapparent redundancy is unusual; while MtrF can functionallyreplace MtrC,[14, 15] the conditions under which the genes forMtrDEF are expressed remain largely unknown. For example,and in contrast to MtrABC, no increase in MtrDEF expres-sion was observed under O2-limited conditions.[16]

The X-ray structures of both outer membrane cyto-chromes, MtrC and MtrF, are now available.[7, 17] Figure 1highlights the intriguing staggered-cross arrangement of theheme-c cofactors. It is hypothesized that heme 10 is the redoxcenter that receives electrons from the periplasmic side of themembrane, while heme 5, the farthest exposed cofactor, is theexit heme that serves as an electron donor for solid acceptorsoutside the cell.[17, 18] On the basis of structural and spectro-scopic observations, it was suggested that MtrC/F could bindflavin mononucleotide (FMN) in a pocket close to heme 7 andthat the binding affinity of FMN is regulated by a redox activedisulfide bridge,[7] close to heme 7 (16 c away). Additionally,experiments suggest that MtrF has a less stable associationwith FMN than MtrC.[7] The difference could be explained bya hydrophobic cleft near heme 7 in MtrC, which is absent inMtrF. Despite the emergence of structural information andthe environmental and biotechnological significance of EET,our understanding of how electron transport (ET) proceedsthrough the multihemes chains of MtrC and MtrF remainsincomplete. This knowledge gap motivated the presentcomparative evaluation of the thermodynamics and ETmechanisms in the two Mtr conduits.

Previous theoretical studies focused on ET in MtrF.[19–21]

Blumberger and co-workers considered the case of a singleelectron hopping through the system; that is, assuming onlyone heme is reduced at a time.[19,20, 22] Ishikita and co-workers

[*] Dr. A. Barrozo, Prof. A. I. KrylovDepartment of Chemistry, University of Southern CaliforniaLos Angeles, CA 90089 (USA)E-mail: krylov@usc.edu

Prof. M. Y. El-NaggarDepartment of Physics, University of Southern CaliforniaLos Angeles, CA 90089 (USA)andDepartment of Chemistry, University of Southern CaliforniaLos Angeles, CA 90089 (USA)andDepartment of Biological Sciences, University of Southern CaliforniaLos Angeles, CA 90089 (USA)

Supporting information and the ORCID identification number(s) forthe author(s) of this article can be found under:https://doi.org/10.1002/anie.201800294.

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took a step further and considered a hole-hopping mechanismwhere a single heme is oxidized at a time and the rest arereduced.[21] In both studies, the computed redox potentialsrequired empirical shifts to account for the energetics of theactual ionization process which requires explicit electronicstructure calculation: @1.567 eV[19] and @0.22 eV[21] added tothe half-redox free energy for heme-c in solution. The twostudies report distinctly different free-energy landscapes: oneshows a path with very low driving force between the terminalhemes,[19] whereas the other shows a clear downhill process.[21]

While shifting the computed potentials by the same value hasno effect on the differences of the redox potentials betweenthe hemes, the empirical shifts make it difficult to elucidatethe central detail of the free-energy landscape, which governsthe ET mechanism, that is, what is the most likely overalloxidation state of the entire system.

We report calculations of electrochemical properties ofMtrC and MtrF in three regimes: electron hopping, in whichonly a single heme is reduced; hole hopping, in which all butone heme are reduced; and the third scenario, a singleelectron hopping with heme 7 reduced. We examine the latterregime because heme 7 has the highest redox potential of theten hemes, as predicted by our calculation, in agreement withprevious work.[19] We calculated redox properties using thelinear response approximation (LRA) within a QM/MMscheme.[23] Using quantum-mechanical potentials instead ofclassic force fields improves the predictive power of thecalculations and allows one to compute absolute values of theredox potentials that can be directly compared to theexperimental measurements.[24, 25] The uncertainties in thesecalculations are due to Gibbs free energy of the referenceelectrode and approximation of Born correction. We used

DG(SHE) = 4.281 eV, which is considered to be the bestestimate[26, 27] (see the Supporting Information). For the hole-hopping regime, our method yielded redox potentials close tothe range determined by protein film voltammetry (PFV).[5,17]

Importantly, in the regime of single electron hopping, thecomputed redox potentials for all 10 hemes are outside therange of the PFV experiments, providing a strong argumentagainst this mechanism, at least under the isolated andelectrode-bound PFV conditions. When we add a secondelectron at heme 7, the calculated redox potentials fall withinthe experimental range. The magnitude of the computedredox shifts in different regimes agrees well with the differ-ence of 250 mV observed in live bacteria at differentconditions and spectroscopically linked to changes in theoxidation state of the cytochromes.[28] In agreement with thestudies on MtrF by Blumberger and co-workers,[19] ourcalculations show no overall driving force across the terminalhemes in all three regimes.

MtrC and MtrF feature similar arrangements of hemecofactors and both have a disulfide bridge at an equivalentposition.[7, 17] A plot of the electrostatic potential in MtrC andMtrF is shown in the Supporting Information, Figure S3. Inboth systems there are several positively charged residuesnear hemes 2 and 7. MtrC features particularly strong positivepotential near heme 2 due to the presence of five lysineresidues within 6 c of the cofactor, whereas MtrF has onlya single lysine in this range. Positive charges near the hemeslower the redox potentials. Of the two views in the SupportingInformation, Figure S3, the top one shows the surface wherethe hemes are the closest, and thus the electrostatic inter-actions from the residues in this surface are stronger.

Figure 2 shows the redox free-energy landscape for MtrCand MtrF in three distinct electron-conducting regimes:1) electron hopping in a fully oxidized system; 2) electronhopping, with heme 7 always reduced and the remaininghemes oxidized; and 3) hole hopping. The average redoxpotential changes by about 0.2 V between (1) and (2), and byabout 0.3 V between (1) and (3); see the SupportingInformation, Table S2. Of the three regimes, electron hoppingresults in the redox potentials that deviate the most from thePFV experimental data (two ranges reported for MtrC: 0 to@450 mV[5] and@25 to@325 mV;[29] the range for MtrF is@46to @312 mV[17]), whereas the redox potentials correspondingto the other two regimes fit better within the range for bothsystems. This suggests that, at least under PFV conditionswhere isolated proteins interact with electrodes, electron flowthrough MtrC/MtrF most likely proceeds with more thana single reduced heme, as in scenarios (2) and (3). Ourcalculations do not rule out the single electron-hopping case.In fact, recent whole cell cyclic and differential pulsevoltammetry experiments yielded more positive values ofthe redox potentials for outer membrane cytochromescompared to PFV (ca. 150 to 300 mV).[9, 30] Owing to its highreduction potential, heme 7 is likely to act as an electron sink.Once heme 7 is reduced, electron transfer back to heme 6 isslow. With FMN (a strong electron acceptor) bound close toheme 7, the cofactor is likely to become oxidized. Ouranalysis suggests that in vivo, where FMN is secreted by thecell, the Mtr proteins conduct in a regime with only one or two

Figure 1. Ten heme-c cofactors arranged in staggered cross in MtrCand MtrF. The current model used to describe the electron flowthrough these two systems for MtrF (PDB ID: 3PMQ[17]) is shown.Yellow spheres denote the location of a redox-active disulfide bridgeimplicated in FMN binding. MtrC shares similar arrangement ofcofactors and position of disulfide bridge.[7]

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electrons flowing through the system at a time. In contrast,under PFV experiments, where the redox potentials are allnegative, one would expect several electrons flowing throughthe system. The computed shift in the absolute values of theredox potentials upon reduction of heme 7 (or other hemes, asin hole hopping regime) is consistent with the results of thedirect spectroscopic measurements of the hemes redox statesin live bacteria;[28] this study reported the difference of about250 mV between the bacteria attached to the electrodes, thatis, at the conditions of a continues electron sink, and thebacteria suspended in the solution. This value is very close toour computed average change in the hemesQ redox potentialsbetween electron hopping regime and when one or morehemes are reduced (ca. 200–350 mV; Supporting Information,Table S2). The absolute values of the redox potential in thelatter case were found to be similar to the redox values ofpurified proteins. On the basis of UV/Vis spectroscopy, theredox state of the cytochromes of the electrode-attachedbacteria was assigned to the oxidized state of the hemes.[28]

Our computed shifts in the redox potentials of theindividual hemes in different regimes point out to a complexnature of heme-to-heme interactions. As discussed in theSupporting Information, the effect of the change of the redoxstate of heme 7 on other hemes cannot be described bya simple electrostatic model, in contrast to the case oftetraheme cytochromes.[31] Our analysis suggests that theinteraction between the hemes includes both the directelectrostatic component and an indirect contribution due tothe variations in structural rearrangements around the hemesinduced by the changes in the redox states of other hemes; thelatter is akin to allosteric effects and is responsible for

apparent lack of correlation of the redox shifts with thedistance between the hemes.

We also note that regardless of the conducting regime, thenet driving force between hemes 10 and 5 is small, inagreement with previous calculations in MtrF.[19] Such a sym-metric energy landscape would allow Mtr proteins to facilitatenot only outward EET, but also the reverse process of inwardEET. This reverse process, where electrons are injected intocells from electrodes, has been demonstrated in S. oneiden-sis[32] and linked to energy gain by the cells.[33]

On the basis of kinetic Monte Carlo model with thereported transfer rates,[20] it was proposed[34] that the transientnumber of electrons in the system is controlled by the rates ofincoming and outgoing electron flux. Our study shows thatthe overall redox potential landscape shifts significantly asa function of the number of reduced hemes in the system. It islikely that the redox state of the protein depends on theenvironment, for example, isolated vs. cell-associated proteinshave different redox potential signatures.[5, 9, 17, 28] Under thePFV experimental conditions, the injection limit can bereached, because the electrode can be tuned to give anarbitrary reduction force. The protein could change itsoxidation state from a single electron to a hole-hoppingregime and back, meaning that the electrode potential maycontrol the redox state of the protein. In contrast, in cellvoltammetry experiments, one is limited by metabolism andultimately the ejection to the electrode controls the preciseredox state of the protein. Studies using cell voltammetry[9,28]

show higher redox potentials than PFV,[5,17] which could beexplained by comparing the single electron hopping scenariowith the two other cases.

If the trends in the shifts of the overall free energy profileas a function of number of reduced hemes hold, the scenariosinvolving three to seven hemes reduced in MtrC/F would alsofit within PFV experiment range. However, the free energylandscape may change and a complete characterizationrequires the assessment of how different oxidation states ofthe system affect the redox potentials of every heme. Becauseof the high costs of full calculations, we limited our study toonly the above three cases.

The hemes in MtrF and MtrC are aligned similarly,[7,17]

with the distances between cofactors differing by less than 1 cbetween the two structures (with the exception of a fewrelative distances to heme 7). Using the reorganizationenergies (Supporting Information, Tables S3 and S4) andsite energies (Table 1) from our calculations, we estimated ETrates for MtrC and MtrF by assuming that the electroniccouplings calculated by Blumberger and co-workers betweenhemes[20] are similar. This is a reasonable assumption based onthe similarities in inter-heme distances between the twostructures (Supporting Information, Table S6). Within theresolution of our calculations, the rate-limiting steps in MtrCand MtrF are comparable, with differences of less than oneorder of magnitude (Supporting Information, Tables S7 andS8). This result suggests that EET is not the main factordetermining higher expression of MtrABC relative toMtrDEF in anoxic environments;[16] however, more precisecalculations are necessary to confidently establish whetherthe two proteins differ in their EET efficiency.

Figure 2. Free-energy landscape of MtrC/F in three different electronconductance regimes. All values are vs. SHE (4.281 V).

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A quantitative characterization of the electrochemicalproperties of a system with multiple redox centers requiresthe determination of distinct oxidation states of the protein,ranging from only one cofactor reduced to the other extreme,where only a single site is oxidized. Our work points to theimportance of electrostatics in multiple-cofactor proteins andillustrates the effect of different electron populations on theenergetics of the redox sites. Our first-principle basedcomputational protocol allows us to discriminate betweendifferent ET regimes by assessing whether or not distinctoxidation states result in the free energy potentials that fallwithin the experimental range. Quantifying the impact ofdifferent electronic populations is a prerequisite for describ-ing the mechanism of ET through the entire system.

The range of the free energy landscape of the S. oneidensisMR-1 outer membrane cytochromes depends on the numberof reduced hemes. Similarly, the redox potential window involtammetry experiments depends strongly on the environ-ment, that is, at isolated electrode-bound conditions of PFVversus in vivo and in bacteria suspended in solution orattached to the electrodes. Comparison between the differentelectron conductance regimes and experimental observationsallows us to conclude that MtrC/F have different electronpopulation according to their environment. Additionally,comparative analysis of the MtrC and MtrF decahemecytochromes suggests that the two systems have comparableET rates of the rate-limiting steps.

Acknowledgements

We are grateful to Profs. Anatoly Kolomeiskii (Rice), AriehWarshel (USC), and Aiichiro Nakano (USC) for stimulatingdiscussions and to Prof. Christopher Cramer (Minnesota) forilluminating analysis of the current status of DG(SHE). Wealso thank the anonymous reviewers whose commentsimproved the manuscript. M.Y.E.-N. acknowledges supportby AFOSR PECASE award FA955014-1-0294. A.I.K.acknowledges support by the U.S. National Science Founda-tion (No. CHE-1566428) and the Extreme Science andEngineering Discovery Environment (XSEDE)[35] supportedby the National Science Foundation grant number ACI-1548562 for access to computational resources. We also

acknowledge the High-Performance Computing Center atUSC.

Conflict of interest

The authors declare no conflict of interest.

Keywords: bacterial nanowires · bioelectronics · cytochromes ·extracellular electron transfer · respiration

How to cite: Angew. Chem. Int. Ed. 2018, 57, 6805–6809Angew. Chem. 2018, 130, 6921–6925

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Table 1: Redox potentials for all the heme cofactors from MtrC and MtrFfor the three different electron conductance regimes studied.[a]

Heme Electron Hopping Hole Hopping Heme 7 ReducedMtrC MtrF MtrC MtrF MtrC MtrF

1 0.045 @0.030 @0.313 @0.281 @0.110 @0.2322 0.178 0.065 @0.217 @0.212 0.070 @0.0823 0.060 @0.015 @0.324 @0.401 @0.258 @0.3374 @0.173 @0.094 @0.304 @0.449 @0.227 @0.3235 0.081 0.045 @0.277 @0.205 @0.142 @0.0876 0.086 0.018 @0.392 @0.343 @0.247 @0.2897 0.113 0.140 @0.257 @0.226 – –8 @0.067 0.013 @0.510 @0.503 @0.279 @0.2119 0.087 0.016 @0.192 @0.180 @0.047 @0.26210 0.032 @0.054 @0.316 @0.190 @0.196 @0.104

[a] All values are in eV vs. SHE (4.281 V).

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Manuscript received: January 9, 2018Revised manuscript received: March 28, 2018Accepted manuscript online: April 17, 2018Version of record online: May 2, 2018

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