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A Voltammetric Study of Nitrogenase Catalysis Using Electron Transfer Mediators Artavazd Badalyan, Zhiyong Yang, and Lance C. Seefeldt ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04290 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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ACS Catalysis

A Voltammetric Study of Nitrogenase Catalysis Using Electron Transfer Mediators Artavazd Badalyan,*,1 Zhi-Yong Yang,1 Lance C. Seefeldt*,1 1

Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan,

UT 84322, USA

Abstract Nitrogenase catalyzes the reduction of an array of small molecules, including N2 to NH3, by delivering electrons and protons to substrates bound to the active site metal cluster FeMo-cofactor. A challenge in describing the mechanism of nitrogenase-catalyzed reduction reactions is quantifying electron flow through the enzyme to different substrates. In this study, a mediated cyclic voltammetry approach was developed that provides a quantitative analysis of electron flow through nitrogenase. Conditions were optimized to reveal the catalytic reaction rate-limiting step. Analysis of the current response by an electrochemical approach yielded a catalytic rate constant (kcat) of 14 s-1, consistent with earlier studies. The current approach was used to resolve a longstanding conundrum in nitrogenase research, the apparent inhibition of electron flow through nitrogenase with increasing partial pressures of N2. It was demonstrated using this voltammetric approach in the absence of the reductant dithionite that total electron flow through nitrogenase remains constant up to an N2 partial pressure of 1 atm.

Keywords: nitrogenase, inhibition, nitrogen reduction, electrocatalysis, mediator, electron flow

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Introduction: The selective reduction of N2 to NH3 is a major challenge for industrial, biological and synthetic chemistry.1 Major efforts have been focused on the development of heterogeneous2–5 and molecular6–8 catalysts that can reduce N2 (using protons and electrons) under ambient conditions that could provide an alternative to the energy intensive and environmentally burdensome HaberBosch process.9 In nature, the enzyme nitrogenase catalyzes the reduction of N2 to 2 NH3 coupled with the reduction of protons to H2.10,11 For the molybdenum-dependent nitrogenase, this reaction proceeds at a reactive metal cluster, FeMo-cofactor, under mild conditions.12 The Mo-nitrogenase is composed of two component proteins (Figure 1a), called the molybdenum-iron protein (MoFeP) and the iron protein (FeP).13 MoFeP houses two unique cofactors, the electron carrier [8Fe-7S] (P-cluster) and the catalytic [7Fe-9S-1Mo-C-homocitrate] (FeMo-co).14 FeP contains a single [4Fe-4S] cluster and two MgATP binding sites. During the catalytic cycle, Fe protein with two bound MgATP, transiently associates with the MoFe protein.15 While the two proteins are associated, a single electron is passed from the Fe protein into the MoFe protein, ultimately accumulating on the FeMo-co (Figure 1b). The two MgATP molecules are then hydrolyzed, triggering the oxidized Fe protein (with two bound MgADP) to dissociate from the one electron reduced MoFe protein. The released Fe protein is reduced, either by flavodoxin or ferredoxin (in vivo)16,17 or sodium dithionite or reduced methyl viologen (in vitro),18,19 and the two MgADP are replaced by two MgATP, to ready the system for another round of association, electron transfer, ATP hydrolysis, and dissociation. This cycle (called the Fe protein cycle) is repeated 4 times to accumulate 4 electrons and 4 protons on FeMo-co as two bridging hydrides and two protons. It is to the four-electron reduced state (called E4(4H)) that N2 can bind, initiating release of H2 through the reductive elimination of the two hydrides and prompt reduction of the

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ACS Catalysis

bound N2 by two electrons (eqn 1).20–22 Four more electron/proton delivery cycles must be completed to achieve the reduction of the N2 to two ammonia molecules.23 In the absence of N2, hydrides and protons react, and H2 is evolved (eqn 2). N2 + 16 MgATP + 8 H+ + 8 e- → 2 NH3 + H2 + 16 MgADP + 16 Pi

(1)

4 MgATP + 2 H+ + 2 e- → H2 + 4 MgADP + 4 Pi

(2)

Figure 1. (a) Simplified catalytic scheme of in vitro nitrogenase catalysis. (b) Electron transfer between redox cofactors of nitrogenase. Shown is Fe in rust, S in yellow, C in gray, Mo in cyan, and O in red. (c) Electrochemical approach to study electron flow through nitrogenase using a diffusive electron transfer mediator.

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The total flow of electrons through nitrogenase is the sum of all electrons delivered to competing substrates. In order to better understand the mechanism of these competing reduction reactions, there is considerable value in quantifying the total flow of electrons through nitrogenase as well as the distribution of electrons to two different substrates. Good techniques are available for quantifying the various products from the reduction of different substrates. Knowing the total electron flux through nitrogenase is essential to answering some key questions about the mechanism. Earlier, the total number of electrons consumed during nitrogenase catalysis was studied under Ar or N2 using activity assays utilizing sodium dithionite as an electron donor. It was found that the total electron flux through nitrogenase decreased by up to 30% at 1 atm N2 compared to no N2 (only H2 formation).24–26 This finding suggested that N2 was inhibiting electron flow through nitrogenase by creating a new overall reaction rate-limiting step, which in turn would have important mechanistic implications. However, the lack of methods to measure total electron flux through nitrogenase has prevented this observation from being validated with a higher precision approach. Electrochemical techniques have been successfully applied for mechanistic studies of catalytic electron transfer for a number of redox enzymes such as hydrogenase,27–32 glucose oxidase,33,34 and others.35–40 For these systems, a homogeneous redox enzymatic system can be connected to an electrode by a freely diffusing mediator. The catalytic response, i.e. catalytic current, may be analyzed to yield a catalytic rate constant. The use of methyl viologen as an electron transfer mediator for nitrogenase has been demonstrated recently in a bioelectrosynthetic cell for ammonia production41 and earlier in non-electrochemical studies.18,19 However, no kinetic analysis has been performed. A challenge for applying an electrochemical approach to nitrogenase comes from the complexity of the catalytic steps in the nitrogenase reaction cycle (Figure 1c). In this work, we

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couple nitrogenase to an electrode by using electron transfer mediators, develop a voltammetric approach that provides highly accurate quantification of electron flow through nitrogenase and demonstrate the application of the approach to address a long-standing question about N2 inhibition of electron flux through nitrogenase.

Results and Discussion Voltammetric studies of nitrogenase under argon. Cyclic voltammetry was applied to study the catalytic behavior of nitrogenase for proton reduction to H2 using methyl viologen as a mediator. The electrochemical studies were performed in activity buffer containing 100 mM MOPS, pH 7.0, 6.7 mM MgCl2, 5 mM ATP and an ATP regeneration system (30 mM phosphocreatine, 0.2 mg/ml kinase, 1.3 mg/ml bovine serum albumin) that showed no significant background electron flow. The electrochemical behavior of methyl viologen was not affected by activity buffer. Upon addition of nitrogenase (MoFeP/FeP), a catalytic current was observed (Figure 2). The current was dependent on a complete catalytic system, with no catalytic current observed if any one of the components (MoFeP, FeP, ATP or MgCl2) was omitted (see Figure S1).

Figure 2. Voltammetric studies of nitrogenase using an electron transfer mediator under argon. Cyclic voltammograms recorded with solutions of activity buffer, corresponding to 100 mM MOPS, pH 7.0, 6.7 mM MgCl2, 5 mM ATP, 30 mM phosphocreatine, 0.2 mg/ml kinase, 1.3

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mg/ml bovine serum albumin (black), upon addition of 50 µM methyl viologen (red), and then addition of nitrogenase complex (MoFeP:FeP, 1:15, blue). The reaction conditions were optimized to permit the assessment of the catalytic performance of nitrogenase (see Supporting Information). The ATP-regeneration system was optimized so that it did not limit the catalytic performance (Figure S2). The catalytic current exhibited a saturation dependence on FeP concentration (Figure 3a, S3), revealing that the reaction rate at ratios of FeP:MoFeP greater than 10:1 is not limited by [FeP]. Further, kinetic analysis was performed at a scan rate at which the catalytic current (which is proportional to the reaction rate) does not exhibit a scan-rate dependence (5 mVs−1, Figure S4).

Figure 3. Nitrogenase Electrocatalysis under argon. a, Plot of current vs. [FeP] in the presence of 50 µM methyl viologen. b, Plot of current vs. [methyl viologen]0.5 in the presence of 0.4 µM MoFeP and 6 µM FeP. All experiments were performed in activity assay buffer containing 100 mM MOPS, pH 7.0, 6.7 mM MgCl2, 5 mM ATP, 30 mM phosphocreatine, 0.2 mg/ml kinase, 1.3 mg/ml bovine serum albumin (3 mL), at 5 mV/s. The results may be analyzed within the kinetic framework formulated by Savéant for enzymatic reactions (see Materials and Methods section for more details).42 The S-shaped voltammograms indicate that the electrocatalysis is not affected by the diffusion of mediator, protons, and corresponds to the ‘kinetic regime’ in which the catalytic current is limited only by chemical steps involving nitrogenase. Under the optimized conditions (also called high-flux

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conditions), the catalytic current exhibits a half-order dependence on concentration of methyl viologen (see Figures 3b and S5), which corresponds to a zero-order dependence of the chemical reaction rate (i.e., kobs) on C⁰Med. The current measured for the catalytic reaction, icat, is described by

eqn 3 2 𝑜 = 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝐸𝑜 𝐶𝑀𝑒𝑑 2𝑘𝑜𝑏𝑠 1

𝑜 𝑖𝑐𝑎𝑡 = 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝐸𝑜 𝐶𝑀𝑒𝑑 √

(3)

𝑘𝑜𝑏𝑠 where kobs is the observed kinetic constant for substrate reduction, F is Faraday’s constant, A is the surface area of the electrode, C⁰Med is the mediator concentration, DMed is the diffusion constant of the mediator, C⁰E is the nitrogenase concentration, kobs is the observed rate constant. This rate expression accounts for the half-order dependence on mediator concentration (C⁰Med) (Fig. 3b) (eqn 3). 𝑖𝑐𝑎𝑡 1 𝑅𝑇𝐶𝐸𝑜 2𝑘𝑜𝑏𝑠 √ = 𝑜 𝑖𝑝 0.4463 𝐹𝑣𝐶𝑀𝑒𝑑

(4)

The ratio of icat/ip in equation 4, where ip is the peak current of the mediator in the absence of nitrogenase, provides a convenient means to determine kobs (R is the ideal gas constant, T is the reaction temperature, and v is the scan rate). A kobs value determined by this method was 14 s-1 for the proton reduction reaction by nitrogenase using methyl viologen as an electron donor. This value is consistent with kobs obtained in the routine nitrogenase activity assays with sodium dithionite as an electron donor in the presence of an excess of FeP -- kobs, 20°C = 10 s-1 and kobs, 30°C = 25 s-1 (the formal potential of sodium dithionite is -660 mV).43 Electron transfer mediators. The design of the developed system is well suited to test other electron transfer mediators (see Supporting Information for more details). For this, redox couples

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have been chosen in the potential range from -0.2 V to -1 V (vs. NHE). Mediators were tested using the same conditions as for methyl viologen. The results are summarized in Table 1. The mediators with potentials more positive than -0.3 V showed no catalysis in the presence of nitrogenase because of the lack of the driving force to reduce FeP (-0.4 V vs. NHE, measured at pH 8 in the presence of MgATP).44,45 The reduction rates of nitrogenase with mediators (Eo < -0.3 V) are increasing and reach maximum for methyl viologen except for Neutral Red and Co(sepulchrate)3+/2+, where only slow electrocatalysis has been observed. A further increase of driving force by using more potent reductants does not lead to an increase of the electrocatalytic rate. With the mediator with the lowest standard potential, cobaltocene, the catalysis slows down most probably due to the low solubility of the reduced form of the cobalt complex. Efficient mediators for nitrogenase are methyl and ethyl viologens, triquat and carboxylated cobaltocenes. Europium complexes, which have been applied for MoFeP-only studies,46 were not tested because of high background reduction current observed in cyclic voltammograms (data not shown). To conclude, the mediator test reveals a thermodynamic control of electron transfer between mediators and a surface exposed [Fe4S4]-cofactor of FeP. Table 1. Summary of Mediator Performance with Nitrogenase under Argon

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N2 reduction. To study N2 reduction, electrolysis was performed in N2 saturated buffer using methyl viologen as a mediator. NH3 was formed with a Faradaic efficiency of 55% (see Supporting Information), which is consistent with previously reported observations.41 Thus, the total electron flux can be deduced by summing all of the electrons delivered to N2 and H+. Cyclic voltammograms recorded under N2 (Figure 4) did not differ from those obtained in the absence of N2 (under argon, Figure 2). A kobs value determined under N2 was 14 s-1, revealing that the proton reduction reaction (no N2) and N2 reduction reaction proceed with the same rate of electron flux through nitrogenase i.e.

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Figure 4. Effect of N2 on total electron flux. CV’s were recorded under N2 in activity buffer solution, corresponding to 100 mM MOPS, pH 7.0, 6.7 mM MgCl2, 5 mM ATP, 30 mM phosphocreatine, 0.2 mg/ml kinase, 1.3 mg/ml bovine serum albumin (black), upon addition of 50 µM methyl viologen (red), followed by addition of nitrogenase (MoFeP:FeP, 1:15, blue). In green, a catalytic CV in the absence of N2. All experiments were performed at 5 mV/s (3 mL) under anaerobic conditions.

The slow step of nitrogenase catalysis. No change of the catalytic performance under N2 may be considered with the electrochemical insight that the catalytic current dependence on methyl viologen exhibits a half-order dependence, revealing that the reduction of nitrogenase by methyl viologen is not rate-limiting. Combining these observations, the slowest step for both N2 reduction and proton reduction by nitrogenase is consistent with earlier findings that the rate-limiting step is in electron delivery through nitrogenase.16 The rate constant determined by using cyclic voltammetry (14 s-1) is close to the previously reported phosphate release rate of 25-27 s-1 (2 Pi per e-, measured with sodium dithionite). This reaction precedes the dissociation of the MoFeP/FeP complex and has been shown to limit the overall reaction rate.47

Conclusions: This work demonstrates an accurate, direct method to quantify electron delivery to nitrogenase. Nitrogenase was found to accept electrons from low-potential organic molecules and cobalt

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complexes. Analysis of the system revealed that the rate-limiting step was associated with nitrogenase function, and not the electrochemical system. This allowed for the nitrogenase reaction rate-limiting step to be revealed with a deduced overall rate constant of 14 s-1. Importantly, this approach allowed resolution of a long-standing confusion about nitrogenase catalysis, showing that the rate-limiting step remains the same when the enzyme is reducing protons alone or N2, consistent with a step in the nitrogenase electron delivery remaining rate limiting. The current approach allows for highly accurate quantification of electron flow through nitrogenase, which should have application in the study of many aspects of the nitrogenase mechanism.

Experimental Section Reagents and Apparatus. All commercial reagents were obtained from Sigma-Aldrich and used as received unless otherwise noted. Dihydrogen, argon, and dinitrogen were purchased from Air Liquide America Specialty Gases LLC (Plumsteadville, PA). 1-Carboxy-cobaltocenium hexafluorophosphate and 1,1'-dicarboxy-cobaltocenium hexafluorophosphate were obtained from MCAT GmbH (Donaueschingen, Germany). The argon and dinitrogen gases were passed through an activated copper catalyst to remove dioxygen contamination prior to use. A. vinelandii strains DJ995 (wild-type MoFeP protein, UniProtKB P07328, P07329) and DJ884 (wild-type Fe protein, UniProtKB P00459) were grown, and nitrogenase proteins were expressed and purified as previously described.48 Proteins and buffers were handled anaerobically in septum-sealed serum vials under an inert atmosphere (argon or dinitrogen), on a Schlenk vacuum line. The transfer of gases and liquids was done with gas-tight syringes.

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Cyclic voltammetry (CV) measurements were performed on a Palmsens 4 (Utrecht, Netherlands) under Ar or N2 in the anaerobic glove box (MO-M, Vacuum Atmosphere Company, Hawthorne, CA). Voltammograms were measured in aqueous buffer solution (100 mM MOPS, pH 7.0) at room temperature (22-23°C). The conventional three-electrode cell (SVC-2, ALS, Japan) was used with a platinum wire counter electrode and with a glassy carbon (GC) working electrode (diameter 3 mm, BASi, USA or ALS, Japan). Working electrodes were routinely polished with BASi polishing alumina suspension (1 µm), rinsed with water, sonicated and dried under air before use. The potentials were measured with respect to the reference electrode – Saturated Calomel Electrode (SCE, ALS, Japan). Steady-State Proton Reduction Assays. Substrate reduction assays were conducted in 9.4 mL sealed serum vials with a liquid volume of 1 mL in an assay buffer consisting of 6.7 mM MgCl 2, 30 mM phosphocreatine, 5 mM ATP, 0.2 mg/mL creatine phosphokinase, and 1.3 mg/mL BSA in 100 mM MOPS buffer (pH 7.0) with 14.4 mM sodium dithionite (DT). After solutions were made anaerobic, the headspace in the reaction vials was adjusted with argon. The MoFeP protein was then added to a final concentration of 0.4 µM (0.1 mg/mL). Each reaction vial was incubated for 8 minutes at 20°C or 30°C for 8 min after initiation of the reaction by the addition of an excess of Fe protein (6 µM or 8 µM, respectively). Reactions were quenched by the addition of 300 μL of 400 mM EDTA. The product (H2) from different substrate reduction assays were quantified according to a published method.49 Evaluation of Kinetic Parameters. The kinetic scheme for nitrogenase catalysis can be found elsewhere and may be rewritten in the following form: MV = methyl viologen

FeP = iron protein (free or nucleotide-bound)

MoFeP = MoFe protein

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ACS Catalysis

𝐌𝐕 𝟐+ + 𝐞− → 𝐌𝐕 𝟏+

(5)

𝐌𝐕 𝟏+ + 𝐅𝐞𝐏𝐎𝐱 ↔ [𝐌𝐕 𝟏+ ∙ 𝐅𝐞𝐏𝐎𝐱 ] → 𝐌𝐕 𝟐+ + 𝐅𝐞𝐏𝐑𝐞𝐝

(6)

𝐅𝐞𝐏𝐑𝐞𝐝 + 𝟐𝐌𝐠𝐀𝐓𝐏 + 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 → 𝐅𝐞𝐏𝐑𝐞𝐝 ∙ 𝟐𝐌𝐠𝐀𝐓𝐏 ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱

(7)

𝐅𝐞𝐏𝐑𝐞𝐝 ∙ 𝟐𝐌𝐠𝐀𝐓𝐏 ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 → 𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐌𝐠𝐀𝐃𝐏 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐑𝐞𝐝

(8)

𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐌𝐠𝐀𝐃𝐏 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐑𝐞𝐝 + 𝐇 +

(9)

↔ [𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐌𝐠𝐀𝐃𝐏 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐑𝐞𝐝 ∙ 𝐇 + ] 𝟏 → 𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐌𝐠𝐀𝐃𝐏 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 + 𝑯𝟐 𝟐 𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐌𝐠𝐀𝐃𝐏 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 → 𝐅𝐞𝐏𝐎𝐱 + 𝟐𝐌𝐠𝐀𝐃𝐏 + 𝟐𝐏 − + 𝐌𝐨𝐅𝐞𝐏𝐎𝐱

(10)

Further assumptions were taken into consideration, based on the previous reports: the catalytic scheme can be simplified as follows: 1. ATP concentration is saturating, and ATP regeneration is fast (i.e. CATP = const). 2. FeP reduction is fast. Reaction (eq. 6) is not rate limiting. 3. Concentration of protons is much larger than Km of nitrogenase. 4. The reduction of substrate (protons) takes place in the MoFeP/FeP-complex. 5. FeP concentration is saturating. 6. The active catalyst may be considered as a MoFeP/FeP-complex. C°complex = C°MoFeP = C°E. Based on this, the catalytic scheme can be rewritten as follows: 𝐌𝐕 𝟐+ + 𝐞− → 𝐌𝐕 𝟏+ 𝒌𝟐,𝟏 ,𝒌𝟐,−𝟏

𝑬𝟐 + 𝑴𝑽𝟏+ ↔

𝒌𝟏,𝟏 ,𝒌𝟏,−𝟏

𝑬 𝟏 + 𝑯+ ↔

(5) 𝒌𝟐,𝟐

𝑬𝟐 ∙ 𝑴𝑽𝟏+ → 𝑬𝟏 + 𝑴𝑽𝟐+

𝒌∗𝟏,𝟐 𝟏 𝑬𝟏 ∙ 𝑯+ → 𝑬∗𝟐 + 𝑯𝟐 𝟐

(11) (12)

𝒌𝟏,𝟐

𝑬∗𝟐 → 𝑬𝟐 ,

(13)

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where 𝑬𝟐 = 𝐅𝐞𝐏𝐎𝐱 ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 ; 𝑬𝟏 = 𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐑𝐞𝐝; 𝑬∗𝟐 = 𝐅𝐞𝐏𝐎𝐱 ∙ 𝟐𝐏 − ∙ 𝐌𝐨𝐅𝐞𝐏𝐎𝐱 The scheme including equations 5, 11, 12 has been considered by Savéant and coworkers as relevant to enzymatic reactions where the solution-phase chemical reaction is rate-limiting (i.e., electron-transfer to the electrode does not contribute to the reaction rate). Two analytical solutions for this scheme were obtained. One solution described the catalytic current when the mediator reaction is the slowest step, which is not the case of nitrogenase. The second solution is for the substrate reduction as a limiting step. Here, it was not clear if substrate reduction (k*1,2) or Pi release and the following dissociation of nitrogenase complex (k1,2) was rate-limiting. In both cases, the kinetic analysis is the same with k*1,2 or k1,2 being the observed kinetic constants (kobs). In this case, the catalytic current of an S-shaped voltammogram is represented by the following equation: 𝑜 𝑖𝑐𝑎𝑡 = 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝑀𝑒𝑑

2𝐶𝐸𝑜 √ 1 1 1 ( + + ) 𝑘2,2 𝑘𝑜𝑏𝑠 𝑘1 𝐶𝐻𝑜 + /𝐾𝑀, 𝐻 +

(14)

Applying assumptions (2) and (3), the equation can be rewritten:

𝑜 𝑖𝑐𝑎𝑡 = 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝐸𝑜 𝐶𝑀𝑒𝑑 √

2 𝑜 = 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝐸𝑜 𝐶𝑀𝑒𝑑 2𝑘𝑜𝑏𝑠 1

(15)

𝑘𝑜𝑏𝑠 The current for a diffusive species in the absence of catalysis is described by the Randles-Sevcik equation: 𝑛𝐹𝑣𝐷𝑀𝑒𝑑 𝑜 √ 𝑖𝑝 = 0.4463𝐹𝐴𝐶𝑀𝑒𝑑 𝑅𝑇

(16)

The ratio of current under catalytic and noncatalytic condition gives the following expression:

14


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ACS Catalysis

𝑜 𝐹𝐴√𝐷𝑀𝑒𝑑 𝐶𝐸𝑜 𝐶𝑀𝑒𝑑 2𝑘𝑜𝑏𝑠 𝑖𝑐𝑎𝑡 1 𝑅𝑇𝐶𝐸𝑜 2𝑘𝑜𝑏𝑠 √𝐶𝐸𝑜 2𝑘𝑜𝑏𝑠 √ = = = 𝑜 𝑜 𝑖𝑝 0.4463 𝐹𝑣𝐶𝑀𝑒𝑑 𝑛𝐹𝑣𝐶𝑀𝑒𝑑 𝑜 √𝑛𝐹𝑣𝐷𝑀𝑒𝑑 √ 0.4463𝐹𝐴𝐶𝑀𝑒𝑑 0.4463 𝑅𝑇 𝑅𝑇

(17)

kobs can be extracted from this equation: 2

𝑘𝑜𝑏𝑠 = 𝑘1,2 (𝑜𝑟

∗ 𝑘1,2 )

𝑜 𝑖𝑐𝑎𝑡 𝑛𝐹𝑣𝐶𝑀𝑒𝑑 = (0.4463 ) 𝑖𝑝 𝑅𝑇𝐶𝐸𝑜 2

(18)

The derivation of equations was done for one-electron reaction and represents the electron flow through nitrogenase.

Author Information Corresponding Authors * E-mail for A.B.: [email protected] * E-mail for L.C.S.: [email protected] ORCID Artavazd Badalyan: 0000-0002-6933-6181 Zhi-Yong Yang: 0000-0001-8186-9450 Lance Seefeldt: 0000-0002-6457-9504 Author Contributions A.B. conceived the idea of the project and designed the study. Z.Y. isolated and purified MoFeP and FeP. A.B. performed all electrochemical experiments and activity assays and led the data interpretation and analysis in consultation with L.C.S. A.B., Z.Y., and L.C.S. wrote the manuscript. Notes The authors declare no competing ﬁnancial interest. Associated content

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Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: Cyclic voltammetry data for control experiments, optimization of nitrogenase/methyl viologen system, mediator screening, electrolysis data, synthesis and NMR spectrum of triquat (PDF). Acknowledgments Support was provided by the US. Department of Energy, Office of Science, Basic Energy Sciences, Physical Biosciences (DE-SC0010687). References. (1) Chen, J. G.; Crooks, R. M.; Seefeldt, L. C.; Bren, K. L.; Bullock, R. M.; Darensbourg, M. Y.; Holland, P. L.; Hoffman, B.; Janik, M. J.; Jones, A. K.; Kanatzidis, M. G.; King, P.; Lancaster, K. M.; Lymar, S. V.; Pfromm, P.; Schneider, W. F.; Schrock, R. R. Beyond Fossil Fuel–Driven Nitrogen Transformations. Science 2018, 360, eaar6611. (2) Cui, X.; Tang, C.; Zhang, Q. A Review of Electrocatalytic Reduction of Dinitrogen to Ammonia under Ambient Conditions. Adv. Energy Mater. 2018, 8, 1800369. (3) Medford, A. J.; Hatzell, M. C. Photon-Driven Nitrogen Fixation: Current Progress, Thermodynamic Considerations, and Future Outlook. ACS Catal. 2017, 7, 2624–2643. (4) Ham, C. J. M. van der; Koper, M. T. M.; Hetterscheid, D. G. H. Challenges in Reduction of Dinitrogen by Proton and Electron Transfer. Chem. Soc. Rev. 2014, 43, 5183–5191. (5) Singh, A. R.; Rohr, B. A.; Schwalbe, J. A.; Cargnello, M.; Chan, K.; Jaramillo, T. F.; Chorkendorff, I.; Nørskov, J. K. Electrochemical Ammonia Synthesis—The Selectivity Challenge. ACS Catal. 2017, 7, 706–709. (6) MacLeod, K. C.; Holland, P. L. Recent Developments in the Homogeneous Reduction of Dinitrogen by Molybdenum and Iron. Nat. Chem. 2013, 5, 559–565. (7) Nishibayashi, Y. Recent Progress in Transition-Metal-Catalyzed Reduction of Molecular Dinitrogen under Ambient Reaction Conditions. Inorg. Chem. 2015, 54, 9234–9247. (8) Roux, Y.; Duboc, C.; Gennari, M. Molecular Catalysts for N2 Reduction: State of the Art, Mechanism, and Challenges. ChemPhysChem 2017, 18, 2606–2617. (9) Wang, L.; Xia, M.; Wang, H.; Huang, K.; Qian, C.; Maravelias, C. T.; Ozin, G. A. Greening Ammonia toward the Solar Ammonia Refinery. Joule 2018, 2, 1055–1074. (10) Burgess, B. K.; Lowe, D. J. Mechanism of Molybdenum Nitrogenase. Chem. Rev. 1996, 96, 2983–3012. (11) Eady, R. R. Structure-Function Relationships of Alternative Nitrogenases. Chem. Rev. 1996, 96, 3013–3030.

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