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Acceleration of Extracellular Electron Transfer by Alternative Redox Active Molecules to Riboflavin for OuterMembrane Cytochrome C of Shewanella oneidensis MR-1 Yoshihide Tokunou, Kazuhito Hashimoto, and Akihiro Okamoto J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b00349 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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The Journal of Physical Chemistry

Acceleration of Extracellular Electron Transfer by Alternative Redox Active Molecules to Riboflavin for Outer-Membrane Cytochrome C of Shewanella oneidensis MR-1 Yoshihide Tokunou, Kazuhito Hashimoto,* and Akihiro Okamoto*

Department of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

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ABSTRACT

Extracellular electron transfer (EET) between outer membrane c-type cytochromes (OM c-Cyts), of Shewanella oneidensis MR-1 and anodic and cathodic electrodes is markedly enhanced by the presence of riboflavin that operates as a redox active center in OM c-Cyt protein OmcA. Here, to obtain insight into the EET mechanism via bound riboflavin in OmcA, we replaced riboflavin with two flavin-like polycyclic molecules, safranin (Saf) and anthraquinone-1-sulfonate (α-AQS), and examined the influence of the interaction with OmcA and the potential of these redox molecules for the rate of cathodic EET by MR-1 in vivo. The results of electrochemical assays with wild-type and mutant strains of MR-1 lacking OmcA showed that both Saf and α-AQS increased the cathodic current production for fumarate reduction at −0.45 V vs SHE as a bound cofactor in OmcA. Dissociation constant and enhancement factor analyses of Saf and α-AQS suggested that the N(5) atom in the isoalloxazine ring of riboflavin is important for the affinity with OmcA and that a positive redox potential is critical for a high rate of EET. The present results suggest that the molecular design of the redox active center in OM c-Cyts may allow control of the rate of EET in EET-capable bacteria.


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1. INTRODUCTION

Some species of iron-reducing bacteria, such as Shewanella oneidensis MR-1 and Geobacter sulfurreducens, are capable of exchanging electrons with extracellular anodic or cathodic electrodes.1-4 This type of interfacial electron transfer, termed extracellular electron transfer (EET), enables individual microbes to operate as either anodic or cathodic electrode catalysts by coupling intracellular enzymatic reactions with EET. This unique characteristic of iron-reducing bacteria has significant implications for the design and development of emerging bioelectrochemical technologies, such as microbial fuel cells5 and electrode biosynthesis,4, 6 as well as for geochemical mineral cycling,7 bioremediation,8-9 and anaerobic iron corrosion.10-11 For this reason, the molecular-level elucidation of the EET process is critical for understanding and controlling the kinetics of the underlying reactions. In S. oneidensis MR-1, either anodic or cathodic electrons flow through a multi-heme c-type cytochrome complex (MtrCAB-OmcA) located on the surface of the outer membrane.12-14 The rate of interfacial electron transfer mediated by these outer membrane c-type cytochromes (OM c-Cyts) is enhanced by the presence of small redox molecules that act as either shuttles5 or bound cofactors15. Although several electron shuttles have been identified to date,5, 16-17 only riboflavin (RF) and flavin mononucleotide (FMN) are known to function as non-covalently bound redox active centers in OM c-Cyts.18-20 The identification and characterization of alternative cofactor molecules is important for understanding the EET 3 ACS Paragon Plus Environment


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mechanism mediated by flavins bound to OM c-Cyts, particularly the kinetics and molecular interactions between non-covalent flavin cofactors and the binding pocket of MtrC and OmcA protein. Notably, because EET mediated by bound cofactors is estimated to require 10- to 100-fold less redox molecules than EET processes that proceed via redox electron shuttles,16 the identification of other molecules that have the potential to function as redox cofactors in OM c-Cyts is expected to facilitate the control of the rate of EET in various electrochemical applications, such as microbial fuel cells and electrode biosynthesis. In the present study, we examined the potential of compounds other than flavin to function as bound redox cofactors in OM c-Cyts using whole-cell electrochemical assays performed under cathodic conditions. Cathodic conditions were selected because we previously demonstrated that microbial electron uptake is enhanced by RF, which selectively binds to OmcA, but not by FMN.21-22 Although specific lock-and-key interactions are predicted to exist between RF and OmcA , the longer side chain of FMN compared to that of RF may hinder the interaction between FMN and OmcA, as RF and FMN only differ by the presence of a phosphate group in the side chain of FMN (Figure 1). If these structural predictions are correct, flavin-like polycyclic molecules with shorter side chain may have the potential to serve as redox cofactors in OmcA protein. Here, we therefore selected two polycyclic compounds with short side chains, safranin (Saf) and anthraquinone-1-sulfonate (α-AQS) (Figure 1), and compared their EET kinetics and binding affinity for OM c-Cyts to obtain


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valuable thermodynamic and structural insight into the molecular interaction between OmcA and riboflavin, as well as the design of cofactors for controlling the rate of EET for both anodic and cathodic reactions.

2. EXPERIMENTAL SECTION 2-1. Bacterial preparation S. oneidensis MR-1 was grown aerobically in 15 mL Luria-Bertani (LB) medium (20 g L-1, Becton Dickinson, Sparks, MD, USA) at 303 K for 24 h. The culture was then centrifuged at 6,000 × g for 10 min, and the resultant cell pellet was resuspended in 15 mL defined medium (DM) supplemented with 10 mM lactate as the sole carbon source.23 The cells were further cultivated aerobically at 303 K for 12 h. After centrifugation at 6,000 × g for 10 min, the resultant cell pellet was washed once with DM prior to use in electrochemical experiments. Mutant strains deficient in the genes encoding either MtrC or OmcA were previously constructed via allele replacement using a two-step homologous recombination method.24 2-2. Electrode-immobilized biofilm construction and electrochemical measurement Electrochemical experiments were conducted under cathodic conditions after the formation of a monolayer biofilm in a single chamber, three-electrode system, as described in previous reports.15,

25

The three-electrode system consisted of an indium tin-doped oxide (ITO)

substrate (surface area of 3.1 cm2) as the working electrode at the bottom of the chamber, and



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Ag/AgCl (KCl saturated) and a platinum wire, which were used as reference and counter electrodes, respectively. Four milliliters of DM containing lactate (10 mM) was deaerated by bubbling with N2 and added into the electrochemical cell as an electrolyte. The reactor was maintained at a temperature of 303 K and was not agitated during the measurements. A cell suspension of S. oneidensis MR-1 in DM with an optical density at λ = 600 nm (OD600) of 0.1 was cultivated on the ITO electrode, which was poised at a potential of +0.4 V vs. SHE in the presence of lactate as an electron source for 25 h. After confirming the formation of a biofilm by in-situ confocal fluorescence microscopy, the supernatant in the electrochemical cell was replaced with anaerobic DM containing 50 mM fumarate as an electron acceptor, and the electrode potential was shifted to −0.45 V to promote the donation of electrons to microbes. Differential pulse (DP) voltammetry was conducted using an automatic polarization system (VMP3, Bio Logic Co.) and the following conditions: 5.0 mV pulse increments, 50 mV pulse amplitude, 300 ms pulse width, and a 5.0 s pulse period. Charging current subtraction was performed using the open source program SOAS by fitting the baseline from regions sufficiently far from the target reduction peaks and assuming that continual charging current flows throughout the peak region.26 2-3. Calculation of current enhancement factor (β) using dissociation constant (Kd) When the cathodic current (Ic) is proportionally related to the amount of redox cofactor binding, the normalized enhancement factor (β) is defined as follows:


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β =

‫ܿܫ‬ [PL]

For the calculation of β, we estimated the dissociation constant (Kd) for complex formation between the non-covalent redox cofactors and OM c-Cyts using the following equation: Kd =

[P][L] [PL]

where [P] is the concentration of OM c-Cyts, [L] is the concentration of unbound RF, Saf or AQS in solution, and [PL] is the concentration of the OM c-Cyts complex with these moleucles. Kd was estimated from the cathodic current (Ic) and the reduction peak current in DP voltammograms before and after the addition of each redox molecule, as previously described.27 By using these equations, β can be described as follows: β=

ሺ[L] + K d ሻ‫ܿܫ‬ [L]ሺ[P] + [PL]ሻ

To compare β among the different redox molecules, [L] was set as 2 µM and the sum of [P] and [PL] was assumed to be constant in the uniform monolayer biofilm of MR-1.

3. RESULTS AND DISCUSSION

3-1. Saf and α-AQS enhance extracellular electron uptake as redox active centers in OmcA

To determine if Saf and α-AQS can function as redox cofactors in OM c-Cyts and enhance the rate of EET, we examined cathodic current production for fumarate reduction28 in the 7 ACS Paragon Plus Environment


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presence of Saf and α-AQS at a concentration of 2 µM, because EET mediated by shuttling redox mediators requires 10- to 100-fold higher concentrations.16 In the presence of 50 mM fumarate as the sole electron acceptor, a S. oneidensis MR-1 biofilm generated a cathodic current (Ic) of approximately 2.5 µA at −0.45 V (vs. SHE). However, upon the addition of 2 µM Saf, an immediate increase in Ic comparable to that resulting from the addition of 2 µM RF was detected (Figure 2). Although the increase was markedly less compared to the systems supplemented with Saf or RF, the addition of α-AQS also led to an enhancement in Ic. These results suggest that Saf and α-AQS enhance the rate of electron uptake by OM c-Cyts by serving as redox active centers, as has been demonstrated for RF.21

To examine the electrochemical properties of Saf and α-AQS in the monolayer biofilm of MR-1, DP voltammetry was conducted during the course of the Ic measurement. After the addition of 4 µM Saf to the electrochemical reactor containing an MR-1 monolayer biofilm, the peak cathodic current was observed at −0.45 V vs SHE (I-0.45) (Figure 3a) and increased with increasing Saf concentration (Figure S1a, Supporting Information), indicating that the cathodic peak at −0.45 V (I-0.45) is assignable to the redox reaction mediated by Saf. The half-width potential (∆Ep/2) for I-0.45 was approximately 190 mV (Figure 3a), which is close to that reported for the one-electron redox reaction of bound RF in OmcA under cathodic conditions.21 In contrast, the two-electron redox reaction of unbound Saf in a cell-free system has ∆Ep/2 and Ep values of approximately 75 mV and −0.30 V, respectively (Figure 3a). These 8 ACS Paragon Plus Environment

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redox profiles strongly suggest that Saf mediates one-electron redox reactions in MR-1 biofilms, similar to bound RF cofactors in OmcA. In addition, DP voltammograms of α-AQS in the presence or absence of biofilm showed redox profiles characteristic of one- or two-electron redox reactions, respectively (Figure 3b and Figure S1b). These results indicate that both Saf and α-AQS are able to mediate one-electron reactions in MR-1 biofilms, as has been reported for RF as a bound cofactor in OmcA, and further suggest that short side chains in polycyclic redox molecules are important for their functioning as redox active centers in OmcA.

To examine the interaction between OM c-Cyts and Saf or α-AQS in biofilms, we used mutant strains of MR-1 that lack either OmcA (∆omcA) or MtrC (∆mtrC). In the DP voltammogram for the ∆omcA biofilm in the presence of Saf, an approximately 90-mV positive shift in the redox signal compared to that for the wild-type (WT) system was observed (Figure 3a), indicating that OmcA interacts with Saf in the biofilm. However, the negative shift (~60 mV) in the peak potential of unbound Saf suggests that Saf interacts with not only OmcA, but also other OM c-Cyts, such as MtrC, in MR-1 cells. In contrast, an identical peak potential and peak width were observed in the DP voltammograms for α-AQS in the presence or absence of ∆omcA biofilms (Fig. 3b). Observed peak potential and width demonstrate that α-AQS mediates 2-electron redox reaction as soluble state even with ∆omcA cells, suggesting that α-AQS specifically associates with OmcA protein. In addition, the Ic of 9 ACS Paragon Plus Environment


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the ∆mtrC monolayer biofilm in the presence of Saf was approximately 15% lower than that for the WT biofilm, whereas comparable values between ∆mtrC and WT were detected in the systems supplemented with α-AQS (Figure S2). Taken together, these results strongly suggest that Saf functions as a bound cofactor in OM c-Cyts of MR-1, mainly in OmcA, but also in MtrC, and that α-AQS specifically interacts with OmcA to enhance microbial cathodic electron uptake.

We also examined the contribution of the cathodic reaction of Saf and α-AQS to Ic in the presence of the MR-1 biofilm by plotting Ic against I-0.45 and I-0.55, which correspond to the relative amount of Saf and α-AQS, respectively. In the course of current generation, we examined the effects of several different concentrations of Saf and α-AQS in the electrochemical system and performed DP voltammetry at each concentration (Figure S3). Ic exhibited a clear negative correlation with both I-0.45 and I-0.55, giving square of the correlation coefficient values of 0.997 and 0.995, respectively (Figure 3c). These results indicate that complex formation of either Saf or α-AQS with OM c-Cyt proteins enhances the rate of electron uptake from the electrode. In addition, these results also suggest that the rate-determining factor for Ic is the number of polycyclic molecules bound to OM c-Cyts within biofilms formed at the electrode surface under the present experimental conditions.


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3-2. Quantitative comparison of binding affinity to OM c-Cyts and EET kinetics for RF, Saf and α-AQS

To obtain insight into the factors controlling the interaction between OM c-Cyts and the redox molecules, RF, Saf and α-AQS, we next compared the dissociation constant (Kd) between OM c-Cyts and each polycyclic redox molecule based on the observed concentration dependency for the peak currents in DP voltammograms, as described in Experimental Section 2-3 (Figure 4a, inset). Although Saf showed a comparable Kd with RF, α-AQS had a significantly lower affinity towards OM c-Cyts. We initially speculated that the length of side chain from polycyclic backbone is a critical factor determining affinity towards OmcA; however, because the side chain of both Saf and α-AQS are shorter than that of RF, other factors also influence their binding affinity. We previously reported that the redox potential of bound flavin and the ionic strength dependency of Kd are similar for bacterial flavodoxins.29 Crystal structure analysis of flavodoxins has shown that protonation of nitrogen atom at position 5 (N(5)) in the isoalloxazine ring (Figure 4b) is critical for stabilizing one-electron reduced semiquinone cofactors.30 As shown in the molecular structures presented Figure 1, Saf contains a nitrogen atom in the same position of the three-ring polycyclic backbone as RF. As this nitrogen atom is not found in α-AQS, the significantly lower affinity of α-AQS compared to RF and Saf might be due to the absence of this nitrogen atom, which may be required for stabilizing the one-electron reduced state of 11 ACS Paragon Plus Environment


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these redox cofactors in OmcA. In addition, the polarity of the binding molecules might be critical for their affinity with OmcA, because α-AQS with the highest Kd has the strongest electron withdrawing substituent, sulfonic acid group, of the three molecules.

We also compared the enhancement factor (β), EET kinetics among single molecules of RF, Saf and α-AQS, based on the Ic and Kd for each molecule as described in Experimental Section 2-3. Because the molecules with a more positive redox potential showed higher β values (the β for Saf and α-AQS were approximately 60% and 14%, respectively, of the β for RF) (Figure 4a), it appears that electron transfer from the electrode to these redox molecules is the slowest reaction for cathodic fumarate reduction. This speculation is in good agreement with the observed linear relationship between Ic and reduction peak current in DP voltammograms of MR-1 biofilms in the presence of these redox molecules (I-0.45 or I-0.55) (Figure 3c), because these data suggest that the reduction rate of these molecules determines the rate of cathodic fumarate reduction. Therefore, it can be expected that polycyclic compounds with higher redox potentials than RF have the ability to enhance the rate of microbial electron uptake. As many types of polycyclic compounds have the above-mentioned properties, further in-vivo studies examining whether other molecules can accelerate microbial EET in S. oneidensis MR-1 are warranted. In addition, the molecular interactions between RF and OmcA have not been completely resolved and also require further biochemical exploration in-vitro systems.31-32 12 ACS Paragon Plus Environment

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4. Conclusions

In this work, we demonstrated that two polycyclic compounds bearing a short side chain, Saf and α-AQS, accelerate extracellular electron uptake by S. oneidensis MR-1 by functioning as bound redox cofactors in the OM c-Cyt protein, OmcA. This finding indicates that the binding site in OmcA for RF is sufficiently flexible to accept other small molecules. In addition, the data for Kd suggest that the nitrogen atom in the polycyclic backbone is important for the affinity of non-covalent redox cofactors towards OM c-Cyts, a property that may be utilized as a strategy for controlling the rate of EET. Thus, the findings from this study provide a novel approach for designing electrocatalysts that mediate electron transfer between microbes and solid substrates for not only cathodic, but also anodic reactions. Furthermore, because the flavin-binding pocket in OM c-Cyts is also found in another model iron-reducing bacterium, G. sulfurreducens,33 this approach may also be applicable to processes involving

G. sulfurreducens, which is an important species in industrial

bioelechemical reactors.34



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ASSOCIATED CONTENT

Supporting Information.

Differential pulse (DP) voltammograms for monolayer biofilms of S. oneidensis MR-1, time course for cathodic current production (Ic) from a biofilm of S. oneidensis MR-1, and cathodic current production (Ic) from biofilms of WT, ∆omcA, and ∆mtrC. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors *(Email: [email protected]). (A.O.).

*(Email: [email protected]). (K.H.).

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests. 14 ACS Paragon Plus Environment

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ACKNOWLEDGMENT This work was financially supported by a Grant-in-Aid (KAKENHI Grant Number 24000010) for Specially Promoted Research from the Japan Society for Promotion of Science (JSPS).

ABBREVIATIONS EET, extracellular electron transfer; c-Cyts, c-type cytochromes; OM, outer membrane; RF, riboflavin; FMN, flavin mononucleotide; Saf, safranin; α-AQS, anthraquinone-1-sulfonate; LB, Luria-Bertani; DM, defined medium; ITO, tin-doped indium oxide; DP, differential pulse.

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a Mechanism for Controlling the Direction of Electron Flow During Extracellular Electron Transfer. Angew. Chem. Int. Ed. 2014, 2014 53, 10988-10991. 22.

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Figure 1. Chemical structure of polycyclic compounds; flavin mononucleotide (FMN), riboflavin (RF), safranin (Saf), and anthraquinone-1-sulfonate (α-AQS). 81x29mm (300 x 300 DPI)

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Figure 2. Time course for cathodic current production (Ic) from the biofilm of S. oneidensis MR-1 in the presence of 50mM fumarate on an ITO electrode at −0.45V (vs. SHE). Arrow indicates the point at which 2µM Saf, α-AQS, or RF was added to the each batch. The same tendency was reproduced in at least three separate experiments. 81x62mm (300 x 300 DPI)



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Figure 3. Baseline-subtracted differential pulse (DP) voltammograms for monolayer biofilm of S. oneidensis MR-1 (Red line), deletion mutant of omcA (Blue line), and cell-free electrolyte (Black line) in the presence of a) Saf and b) α-AQS at a concentration of 4 µM on an ITO electrode surface. The same tendency was reproduced in three separate experiments. c) Plots of the bacterial cathodic current production (Ic) at an electrode potential of −0.45 V (vs. SHE) against the peak current of Saf and α-AQS (Saf: Ep = −0.45 V vs. SHE, α-AQS: Ep = −0.55 V vs. SHE). The squares of the correlation coefficients include the point of origin. 176x44mm (300 x 300 DPI)


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Figure 4. a) The normalized current enhancement factor (β) for Saf, α-AQS, and RF calculated from at least two separate experiments (see Experimental Section 2-3). We used the cathodic current observed t = 14th hour in Figure 2 as Ic, normalizing as β of RF = 1.0. Inset: Dissociation constant (Kd) for each molecule. Standard deviation in the experimental determination of Kd is indicated. b) Chemical structure of RF. Nitrogen atom at position 5 (N(5)) in isoalloxiazine ring is highlighted. 127x54mm (300 x 300 DPI)



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Graphic for table of context 79x44mm (300 x 300 DPI)


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Acceleration of Extracellular Electron Transfer by ... - ACS Publications

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