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

Research Article

Cite This: ACS Catal. 2019, 9, 1366−1372 pubs.acs.org/acscatalysis

A Voltammetric Study of Nitrogenase Catalysis Using Electron Transfer Mediators Artavazd Badalyan,* Zhi-Yong Yang, and Lance C. Seefeldt* Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322, United States

*S Supporting Information

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 −1 catalytic rate constant (kcat)of14s , consistent with earlier studies. The current approach was used to resolve a long-standing 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 fl electron ow through nitrogenase remains constant up to a N2 partial pressure of 1 atm. KEYWORDS: nitrogenase, inhibition, nitrogen reduction, electrocatalysis, mediator, electron flow

■ INTRODUCTION another round of association, electron transfer, ATP hydrolysis, and dissociation. This cycle (called the Fe protein cycle) is The selective reduction of N2 to NH3 is a major challenge for 1 repeated four times to accumulate four electrons and four industrial, biological, and synthetic chemistry. Major efforts 2−5 protons on FeMo-co as two bridging hydrides and two have been focused on the development of heterogeneous − protons. It is to the four-electron-reduced state [called and molecular6 8 catalysts that can reduce N (using protons 2 E4(4H)] that N can bind, initiating release of H through and electrons) under ambient conditions that could provide an 2 2 the reductive elimination of the two hydrides and prompt alternative to the energy intensive and environmentally − reduction of the bound N by two electrons (eq 1).20 22 Four burdensome Haber−Bosch process.9 In nature, the enzyme 2 more electron/proton delivery cycles must be completed to nitrogenase catalyzes the reduction of N2 to 2NH3 coupled 23 10,11 achieve the reduction of the N2 to two ammonia molecules. Downloaded via UTAH STATE UNIV on September 20, 2019 at 19:54:25 (UTC). with the reduction of protons to H2. For the molybdenum- In the absence of N2, hydrides and protons react, and H2 is dependent nitrogenase, this reaction proceeds at a reactive evolved (eq 2). metal cluster, FeMo-cofactor, under mild conditions.12 See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. The Mo-nitrogenase is composed of two component N+++ 16MgATP 8H+− 8e proteins (Figure 1a), called the molybdenum−iron protein 2 13 (MoFeP) and the iron protein (FeP). MoFeP houses two →++2NH32 H 16MgADP + 16P i (1) unique cofactors, the electron carrier [8Fe-7S] (P-cluster) and 14 +− the catalytic [7Fe-9S-1Mo-C-homocitrate] (FeMo-co). FeP 4MgATP++→+ 2H 2e H2i 4MgADP + 4P (2) contains a single [4Fe-4S] cluster and two MgATP binding fl sites. During the catalytic cycle, Fe protein with two bound The total ow of electrons through nitrogenase is the sum of MgATP transiently associates with the MoFe protein.15 While all electrons delivered to competing substrates. In order to the two proteins are associated, a single electron is passed from better understand the mechanism of these competing the Fe protein into the MoFe protein, ultimately accumulating reduction reactions, there is considerable value in quantifying fl on the FeMo-co (Figure 1b). The two MgATP molecules are the total ow of electrons through nitrogenase as well as the ff then hydrolyzed, triggering the oxidized Fe protein (with two distribution of electrons to two di erent substrates. Good bound MgADP) to dissociate from the one-electron-reduced techniques are available for quantifying the various products MoFe protein. The released Fe protein is reduced, either by flavodoxin or ferredoxin (in vivo)16,17 or by sodium dithionite Received: October 24, 2018 or reduced methyl viologen (in vitro),18,19 and the two Revised: December 19, 2018 MgADP are replaced by two MgATP, to ready the system for Published: January 4, 2019

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. from the reduction of different substrates. Knowing the total electrode by a freely diffusing mediator. The catalytic response, fl electron ux through nitrogenase is essential to answering i.e., catalytic current, may be analyzed to yield a catalytic rate some key questions about the mechanism. Earlier, the total constant. The use of methyl viologen as an electron transfer number of electrons consumed during nitrogenase catalysis was studied under Ar or N using activity assays utilizing mediator for nitrogenase has been demonstrated recently in a 2 41 sodium dithionite as an electron donor. It was found that the bioelectrosynthetic cell for ammonia production and earlier total electron flux through nitrogenase decreased by up to 30% in nonelectrochemical studies.18,19 However, no kinetic 24−26 at 1 atm of N2 compared to no N2 (only H2 formation). analysis has been performed. A challenge for applying an fi fl This nding suggested that N2 was inhibiting electron ow electrochemical approach to nitrogenase comes from the through nitrogenase by creating a new overall reaction-rate- limiting step, which in turn would have important mechanistic complexity of the catalytic steps in the nitrogenase reaction implications. However, the lack of methods to measure total cycle (Figure 1c). In this work, we couple nitrogenase to an electron flux through nitrogenase has prevented this observa- electrode by using electron transfer mediators, develop a tion from being validated with a higher precision approach. voltammetric approach that provides highly accurate quanti- Electrochemical techniques have been successfully applied fication of electron flow through nitrogenase, and demonstrate for mechanistic studies of catalytic electron transfer for a − number of redox enzymes, such as hydrogenase,27 32 glucose the application of the approach to address a long-standing 33,34 35−40 fl oxidase, and others. For these systems, a homoge- question about N2 inhibition of electron ux through neous redox enzymatic system can be connected to an nitrogenase.

1367 DOI: 10.1021/acscatal.8b04290 ACS Catal. 2019, 9, 1366−1372 ACS Catalysis Research Article ■ RESULTS AND DISCUSSION optimized so that it did not limit the catalytic performance Voltammetric Studies of Nitrogenase under Argon. (Figure S2, SI). The catalytic current exhibited a saturation Cyclic voltammetry was applied to study the catalytic behavior dependence on FeP concentration [Figures 3a and S3 (SI)], revealing that the reaction rate at ratios of FeP:MoFeP greater of nitrogenase for proton reduction to H2 using methyl viologen as a mediator. The electrochemical studies were than 10:1 is not limited by [FeP]. Further, kinetic analysis was performed in activity buffer containing 100 mM MOPS, pH performed at a scan rate at which the catalytic current (which is proportional to the reaction rate) does not exhibit a scan- 7.0, 6.7 mM MgCl2, 5 mM ATP, and an ATP regeneration −1 system (30 mM phosphocreatine, 0.2 mg/mL kinase, 1.3 mg/ rate dependence (5 mVs , Figure S4, SI). mL bovine serum albumin) that showed no significant The results may be analyzed within the kinetic framework fl formulated by Saveant́ and coworkers for enzymatic reactions background electron ow. The electrochemical behavior of 42 methyl viologen was not affected by activity buffer. Upon (see the Experimental Section for more details). The S- shaped voltammograms indicate that the electrocatalysis is not addition of nitrogenase (MoFeP/FeP), a catalytic current was ff ff observed (Figure 2). The current was dependent on a a ected by the di usion of mediator and 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 conditions), the catalytic current exhibits a half-order dependence on concentration of methyl viologen [see Figures 3b and S5 (SI)], which corresponds to a zero-order dependence of the 0 chemical reaction rate (i.e., kobs)onCMed. The current measured for the catalytic reaction, icat, is described by eq 3

0 0 icat= FA D Med C E CMed2 kobs (3)

where kobs is the observed kinetic constant for substrate reduction, F is Faraday’s constant, A is the surface area of the 0 electrode, CMed is the mediator concentration, DMed is the ﬀ 0 di usion constant of the mediator, CE is the nitrogenase concentration, and kobs is the observed rate constant. This expression accounts for the half-order dependence of the Figure 2. Voltammetric studies of nitrogenase using an electron 0 transfer mediator under argon. Cyclic voltammograms recorded with catalytic current on mediator concentration (CMed)(Figure solutions of activity buﬀer, corresponding to 100 mM MOPS, pH 7.0, 3b) (eq 3). 6.7 mM MgCl2, 5 mM ATP, 30 mM phosphocreatine, 0.2 mg/mL kinase, and 1.3 mg/mL bovine serum albumin (black), upon addition i 1 RTC02 k μ cat = E obs of 50 M methyl viologen (red) and then addition of nitrogenase i 0.4463 0 complex (MoFeP:FeP, 1:15, blue). p FvCMed (4)

The ratio of icat/ip in eq 4, where ip is the peak current of the complete catalytic system, with no catalytic current observed mediator in the absence of nitrogenase, provides a convenient if any one of the components (MoFeP, FeP, ATP, or MgCl2) means to determine kobs (R is the ideal gas constant, T is the was omitted [see Figure S1, Supporting Information (SI)]. reaction temperature, and v is the scan rate). A kobs value The reaction conditions were optimized to permit the determined by this method was 14 s−1 for the proton reduction assessment of the catalytic performance of nitrogenase (see the reaction by nitrogenase using methyl viologen as an electron Supporting Information). The ATP regeneration system was donor. This value is consistent with the kobs obtained in the

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, and 1.3 mg/mL bovine serum albumin (3 mL), at 5 mV/ s.

1368 DOI: 10.1021/acscatal.8b04290 ACS Catal. 2019, 9, 1366−1372 ACS Catalysis Research Article routine nitrogenase activity assays with sodium dithionite as an electron donor in the presence of an excess of FeP: kobs,20°C = −1 −1 10 s and kobs,30°C =25s (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 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

Table 1. Summary of Mediator Performance with Nitrogenase under Argon

° −1 mediator E vs NHE, mV kobs,s ff fl Figure 4. E ect of N2 on total electron ux. CVs were recorded under − a ff cobaltocene 958 5 N2 in activity bu er solution, corresponding to 100 mM MOPS, pH − a 1-COOH-cobaltocene 840 15 7.0, 6.7 mM MgCl2, 5 mM ATP, 30 mM phosphocreatine, 0.2 mg/mL 1,1′-di-COOH-cobaltocene −690 15 kinase, and 1.3 mg/mL bovine serum albumin (black), upon addition sodium dithioniteb −660 10 of 50 μM methyl viologen (red), followed by addition of nitrogenase triquat −580 12c (MoFeP:FeP, 1:15, blue). In green, a catalytic CV in the absence of ethyl viologen −450 13 N2. All experiments were performed at 5 mV/s (3 mL) under methyl viologen −440 14 anaerobic conditions. Co(sepulchrate)3+/2+ −404 ndd neutral red −390 nd N2) and N2 reduction reaction proceed with the same rate of benzyl viologen −361 8 electron flux through nitrogenase. diquat −360 7 The Slow Step of Nitrogenase Catalysis. No change of mediators (see SI)<−300 nd the catalytic performance under N2 may be considered with aNot a S-shaped voltammogram. bDetermined by a nonelectrochem- the electrochemical insight that the catalytic current depend- ical activity assay (see Supporting Information). cCorrected for ence on methyl viologen exhibits a half-order dependence, d background reduction current. nd = not detectable. 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 fi positive than −0.3 V showed no catalysis in the presence of nitrogenase is consistent with earlier ndings that the rate- limiting step is electron delivery through nitrogenase.16 The nitrogenase because of the lack of the driving force to reduce −1 FeP (−0.4 V vs NHE, measured at pH 8 in the presence of rate constant determined by using cyclic voltammetry (14 s ) MgATP).44,45 The reduction rates of nitrogenase with is close to the previously reported phosphate release rate of − −1 − mediators (Eo < −0.3 V) are increasing and reach a maximum 25 27 s (2Pi per e , measured with sodium dithionite). This reaction precedes the dissociation of the MoFeP/FeP complex for methyl viologen except for neutral red and Co- 47 (sepulchrate)3+/2+, where only slow electrocatalysis has been and has been shown to limit the overall reaction rate. observed. A further increase of driving force by using more potent reductants does not lead to an increase of the ■ CONCLUSIONS electrocatalytic rate. With the mediator with the lowest This work demonstrates an accurate, direct method to quantify standard potential, cobaltocene, the catalysis slows down, electron delivery to nitrogenase. Nitrogenase was found to most probably due to the low solubility of the reduced form of accept electrons from low-potential organic molecules and the cobalt complex. Efficient mediators for nitrogenase are cobalt complexes. Analysis of the system revealed that the rate- methyl and ethyl viologens, triquat, and carboxylated limiting step was associated with nitrogenase function, and not cobaltocenes. Europium complexes, which have been applied the electrochemical system. This allowed for the nitrogenase 46 reaction rate-limiting step to be revealed with a deduced for MoFeP-only studies, were not tested because of the high − background reduction current observed in cyclic voltammo- overall rate constant of 14 s 1. Importantly, this approach grams (data not shown). To conclude, the mediator test allowed resolution of a long-standing confusion about reveals a thermodynamic control of electron transfer between nitrogenase catalysis, showing that the rate-limiting step mediators and a surface exposed [Fe4S4]-cofactor of FeP. remains the same when the enzyme is reducing protons N2 Reduction. To study N2 reduction, electrolysis was alone or N2, consistent with a step in the nitrogenase electron ff performed in N2-saturated bu er using methyl viologen as a delivery remaining rate-limiting. The current approach allows ffi fi fl mediator. NH3 was formed with a Faradaic e ciency of 55% for highly accurate quanti cation of electron ow through (see the Supporting Information), which is consistent with nitrogenase, which should have application in the study of previously reported observations.41 Thus, the total electron many aspects of the nitrogenase mechanism. flux can be deduced by summing all of the electrons delivered + ■ EXPERIMENTAL SECTION to N2 and H . Cyclic voltammograms recorded under N2 (Figure 4) did not differ from those obtained in the absence of Reagents and Apparatus. All commercial reagents were N2 (under argon, Figure 2). A kobs value determined under N2 obtained from Sigma-Aldrich and used as received unless was 14 s−1, revealing that the proton reduction reaction (no otherwise noted. Dihydrogen, argon, and dinitrogen were

1369 DOI: 10.1021/acscatal.8b04290 ACS Catal. 2019, 9, 1366−1372 ACS Catalysis Research Article purchased from Air Liquide America Specialty Gases LLC FeP···+ 2MgADP 2P−+ MoFeP H fl Ox Red (Plumsteadville, PA). 1-Carboxy-cobaltocenium hexa uoro- −+ phosphate and 1,1′-dicarboxy-cobaltocenium hexafluorophos- ↔[FePOx · 2MgADP · 2P · MoFeP Red · H ] phate were obtained from MCAT GmbH (Donaueschingen, 1 →·FeP 2MgADP ·· 2P− MoFeP +H Germany). The argon and dinitrogen gases were passed Ox Ox2 2 (9) through an activated copper catalyst to remove dioxygen − contamination prior to use. Azobacter vinelandii strains DJ995 FePOx··· 2MgADP 2P MoFeP Ox (wild-type MoFeP protein, UniProtKB P07328, P07329) and →+ ++− DJ884 (wild-type Fe protein, UniProtKB P00459) were grown, FePOx 2MgADP 2P MoFeP Ox (10) and nitrogenase proteins were expressed and purified as Further assumptions were taken into consideration based on 48 previously described. Proteins and buffers were handled the previous reports: (1) The ATP concentration is saturating, anaerobically in septum-sealed serum vials under an inert and the ATP regeneration is fast (i.e., CATP = const). (2) The atmosphere (argon or dinitrogen), on a Schlenk vacuum line. FeP reduction is fast. Reaction 6 is not rate-limiting. (3) The The transfer of gases and liquids was done with gastight concentration of protons is much larger than the Km of syringes. nitrogenase. (4) The reduction of substrate (protons) takes Cyclic voltammetry (CV) measurements were performed on place in the MoFeP/FeP complex. (5) The FeP concentration a Palmsens 4 (Utrecht, Netherlands) under Ar or N2 in an is saturating. (6) The active catalyst may be considered as a 0 0 0 anaerobic glovebox (MO-M, Vacuum Atmosphere Co., MoFeP/FeP complex; thus, Ccomplex = CMoFeP = CE. Hawthorne, CA). Voltammograms were measured in aqueous On the basis of this, the catalytic scheme can be simplified as buffer solution (100 mM MOPS, pH 7.0) at room temperature MVeMV21+−+→ + (5) (22−23 °C). The conventional three-electrode cell (SVC-2,

ALS) was used with a platinum wire counter electrode and kk2,1, 2,− 1 k 2,2 +←⎯1++⎯⎯⎯⎯⎯⎯⎯ ·1 ⎯→⎯+2+ with a glassy carbon (GC) working electrode (diameter 3 mm, E2 MV E2 MV E1 MV (11) BASi or ALS). Working electrodes were routinely polished * kk1,1, 1,− 1 k 1,2 1 with BASi polishing alumina suspension (1 μm), rinsed with E +←⎯HEHE++⎯⎯⎯⎯⎯⎯ · ⎯→⎯ * + H 1 122 (12) water, sonicated, and dried under air before use. The potentials 2 were measured with respect to a reference electrode, a k * 1,2 saturated calomel electrode (SCE, ALS). E22⎯→⎯ E (13) Steady-State Proton Reduction Assays. Substrate · · −· where E2 = FePOx MoFePOx,E1 = FePOx 2P MoFePRed, and reduction assays were conducted in 9.4 mL sealed serum E* = FeP ·2P−·MoFeP . ff 2 Ox Ox vials with a liquid volume of 1 mL in an assay bu er consisting The scheme including eqs 5, 11, and 12 has been considered of 6.7 mM MgCl2, 30 mM phosphocreatine, 5 mM ATP, 0.2 by Saveant́ and co-workers as relevant to enzymatic reactions mg/mL creatine phosphokinase, and 1.3 mg/mL BSA in 100 where the solution-phase chemical reaction is rate-limiting mM MOPS buffer (pH 7.0) with 14.4 mM sodium dithionite (i.e., electron-transfer to the electrode does not contribute to (DT). After solutions were made anaerobic, the headspace in the reaction rate). Two analytical solutions for this scheme the reaction vials was adjusted with argon. The MoFeP protein were obtained. One solution described the catalytic current was then added to a final concentration of 0.4 μM (0.1 mg/ when the mediator reaction is the slowest step, which is not the mL). Each reaction vial was incubated for 8 min at 20 or 30 °C case of nitrogenase. The second solution is for the substrate after initiation of the reaction by the addition of an excess of Fe reduction as a limiting step. Here, it was not clear if substrate μ * protein (6 or 8 M, respectively). Reactions were quenched by reduction (k1,2)orPi release and the following dissociation of the addition of 300 μL of 400 mM EDTA. The product (H ) nitrogenase complex (k1,2) were rate-limiting. In both cases, 2 * from different substrate reduction assays was quantified the kinetic analysis is the same with k1,2 or k1,2 being the 49 according to a published method. observed kinetic constants (kobs). In this case, the catalytic Evaluation of Kinetic Parameters. The kinetic scheme current of an S-shaped voltammogram is represented by the for nitrogenase catalysis can be found elsewhere and may be following equation: rewritten in the following form, where MV = methyl viologen, 0 FeP = iron protein (free or nucleotide-bound), and MoFeP = 0 2CE icat= FA D Med C Med MoFe protein: 11++ 1 kkkCK0 / + 2,2 obs 1 H+ M,H (14) MVeMV21+−+→ + (5) Applying assumptions 2 and 3, the equation can be rewritten: ji zy 1+++1 2 j z MV+↔[·]→+ FePOx MV FePOx MV FePRed (6) k { 0 0 2 0 0 icat==FA D Med C E CMed 1 FA DMed C E CMed2 kobs k FePRed++ 2MgATP MoFeP Ox obs (15) →·FeP 2MgATP · MoFeP (7) Red Ox The current for a diffusive species in the absence of catalysis is described by the Randles−Sevcik equation:

FePRed·· 2MgATP MoFeP Ox 0 nFvDMed − ipMed= 0.4463FAC →·FePOx 2MgADP ·· 2P MoFeP Red (8) RT (16)

1370 DOI: 10.1021/acscatal.8b04290 ACS Catal. 2019, 9, 1366−1372 ACS Catalysis Research Article

The ratio of current under catalytic and noncatalytic (4) van der Ham, C. J. M.; Koper, M. T. M.; Hetterscheid, D. G. H. conditions gives the following expression: Challenges in Reduction of Dinitrogen by Proton and Electron Transfer. Chem. Soc. Rev. 2014, 43, 5183−5191. 0 0 0 (5) Singh, A. R.; Rohr, B. A.; Schwalbe, J. A.; Cargnello, M.; Chan, icat FA DMed C E CMed2 kobs CkE2 obs == K.; Jaramillo, T. F.; Chorkendorff, I.; Nørskov, J. K. Electrochemical 0 nFvD 0 ip 0.4463FAC Med nFvCMed Ammonia SynthesisThe Selectivity Challenge. ACS Catal. 2017, 7, Med RT 0.4463 RT 706−709. 0 (6) MacLeod, K. C.; Holland, P. L. Recent Developments in the 1 RTCE2 kobs = 0 Homogeneous Reduction of Dinitrogen by Molybdenum and Iron. 0.4463 nFvCMed (17) Nat. Chem. 2013, 5, 559−565. (7) Nishibayashi, Y. Recent Progress in Transition-Metal-Catalyzed kobs can be extracted from this equation: Reduction of Molecular Dinitrogen under Ambient Reaction 2 − 0 Conditions. Inorg. Chem. 2015, 54, 9234 9247. i nFvC (8) Roux, Y.; Duboc, C.; Gennari, M. Molecular Catalysts for N kkk= (or* )= 0.4463 cat Med 2 obs 1,2 1,2 0 Reduction: State of the Art, Mechanism, and Challenges. ip RTC 2 E (18) ChemPhysChem 2017, 18, 2606−2617. The derivation of equationsi wasy done for a one-electron (9) Wang, L.; Xia, M.; Wang, H.; Huang, K.; Qian, C.; Maravelias, j z reaction (n = 1) and representsj thez electron flow through C. T.; Ozin, G. A. Greening Ammonia toward the Solar Ammonia j z − nitrogenase. k { Refinery. Joule 2018, 2, 1055 1074. (10) Burgess, B. K.; Lowe, D. J. Mechanism of Molybdenum − ■ ASSOCIATED CONTENT Nitrogenase. Chem. Rev. 1996, 96, 2983 3012. (11) Eady, R. R. Structure-Function Relationships of Alternative *S Supporting Information Nitrogenases. Chem. Rev. 1996, 96, 3013−3030. The Supporting Information is available free of charge on the (12) Hoffman, B. M.; Lukoyanov, D.; Yang, Z.-Y.; Dean, D. R.; ACS Publications website at DOI: 10.1021/acscatal.8b04290. Seefeldt, L. C. Mechanism of Nitrogen Fixation by Nitrogenase: The − Cyclic voltammetry data for control experiments, Next Stage. Chem. Rev. 2014, 114, 4041 4062. optimization of the nitrogenase/methyl viologen system, (13) Tezcan, F. A.; Kaiser, J. T.; Mustafi, D.; Walton, M. Y.; Howard, J. B.; Rees, D. C. Nitrogenase Complexes: Multiple Docking mediator screening, electrolysis data, and synthesis and Sites for a Nucleotide Switch Protein. Science 2005, 309, 1377−1380. NMR spectrum of triquat (PDF) (14) Spatzal, T.; Aksoyoglu, M.; Zhang, L.; Andrade, S. L. A.; Schleicher, E.; Weber, S.; Rees, D. C.; Einsle, O. Evidence for ■ AUTHOR INFORMATION Interstitial Carbon in Nitrogenase FeMo Cofactor. Science 2011, 334, Corresponding Authors 940−940. *A.B. e-mail: [email protected]. (15) Seefeldt, L. C.; Hoffman, B. M.; Peters, J. W.; Raugei, S.; *L.C.S. e-mail: [email protected]. Beratan, D. N.; Antony, E.; Dean, D. R. Energy Transduction in Nitrogenase. Acc. Chem. Res. 2018, 51, 2179−2186. 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1372 DOI: 10.1021/acscatal.8b04290 ACS Catal. 2019, 9, 1366−1372