On Reversible H2 Loss Upon N2 Binding to Femo-Cofactor of Nitrogenase

On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase

Zhi-Yong Yanga, Nimesh Khadkaa, Dmitriy Lukoyanovb, Brian M. Hoffmanb,1, Dennis R. Deanc,1, and Lance C. Seefeldta,1

aDepartment of Chemistry and Biochemistry, Utah State University, Logan, UT 84322; bDepartments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, IL 60208; and cDepartment of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061

Contributed by Brian M. Hoffman, August 23, 2013 (sent for review July 15, 2013)

Nitrogenase is activated for N2 reduction by the accumulation of obligatory, reversible H2 loss upon N2 binding (5). We consid- four electrons/protons on its active site FeMo-cofactor, yielding ered two models for this process, both built on our character- a state, designated as E4, which contains two iron-bridging hydrides ization of the key E4 state, and tested them against the numerous – – [Fe H Fe]. A central puzzle of nitrogenase function is an apparently constraints imposed by turnover under N2 plus D2 or T2 (5, 9– obligatory formation of one H2 per N2 reduced, which would “waste” 16). In particular, these constraints include the key findings that two reducing equivalents and four ATP. We recently presented during catalytic reduction of N2 (see Scheme S1), a molecule of a draft mechanism for nitrogenase that provides an explanation D2 or T2 will reduce two protons to form two HD or HT without + + for obligatory H2 production. In this model, H2 is produced by re- D /T exchange with solvent, even though neither D2 nor T2 by ductive elimination of the two bridging hydrides of E4 during N2 themselves reacts with nitrogenase during turnover under Ar (5). binding. This process releases H2, yielding N2 bound to FeMo- One model, involving protonation of one of the hydrides to form cofactor that is doubly reduced relative to the resting redox level, H2, and its replacement by N2, was shown to violate the turnover and thereby is activated to promptly generate bound diazene constraints, and was therefore rejected. = (HN NH). This mechanism predicts that during turnover under In the second model, H2 is produced by reductive elimination D2/N2, the reverse reaction of D2 with the N2-bound product of (re) (17–20) of the two bridging hydrides of E4 during N2 binding reductive elimination would generate dideutero-E4 [E4(2D)], which (Fig. 1B and Scheme S1). This yields N2 bound to FeMo-cofactor can relax with loss of HD to the state designated E2, with a single that has been doubly reduced relative to the resting redox level, deuteride bridge [E2(D)]. Neither of these deuterated intermediate and thereby is activated to promptly deliver two electrons plus states could otherwise form in H2O buffer. The predicted E2(D) and the two protons to N2, generating diazene (HN=NH) bound E4(2D) states are here established by intercepting them with the to FeMo-cofactor. The re activation of FeMo-cofactor for N2 nonphysiological substrate acetylene (C2H2) to generate deuterated binding and reduction involving obligatory H2 formation (i) ethylenes (C2H3DandC2H2D2). The demonstration that gaseous provides a compelling rationale for the apparently wasteful evo- + H2/D2 can reduce a substrate other than H with N2 as a cocatalyst lution of H2 and (ii) satisfies the constraints provided by the fi con rms the essential mechanistic role for H2 formation, and hence interplay of N2 and H2 binding. In particular, as schematized in fi a limiting stoichiometry for biological nitrogen xation of eight Fig. 1C and Fig. S1, according to the re mechanism, D2 or T2 can CHEMISTRY electrons/protons, and provides direct experimental support for reverse the N2 binding equilibrium (1, 21), displace N2, generate the reductive elimination mechanism. an E4 intermediate with two [Fe–D/T–Fe], which do not exchange with solvent (5, 11). This intermediate in turn can relax in acetylene reduction | metalloenzyme two steps to form two HD or HT, thereby satisfying these two key constraints. As a result, the re mechanism formed the key-

iological nitrogen ﬁxation—the reduction of dinitrogen (N2) stone of our draft mechanism. — Subsequently, we recognized that this re mechanism for H Bto two ammonia (NH3) molecules is primarily catalyzed 2

by the Mo-dependent nitrogenase. This enzyme comprises an release upon N2 binding (Fig. 1B) makes testable predictions. As BIOCHEMISTRY electron-delivery Fe protein and MoFe protein, which contains fi the active site FeMo-cofactor (Fig. 1A) (1, 2). The nitrogenase Signi cance catalyzed reaction is generally thought to have the limiting stoichiometry shown in Eq. 1 (3), Biological reduction of dinitrogen (N2) to two ammonia (NH3)is catalyzed by the metalloenzyme, nitrogenase. A central aspect + of nitrogenase function is an apparently obligatory formation N2 + 8e + 16ATP + 8H → 2NH3 + H2 + 16ADP + 16Pi: [1] of one H2 per N2 reduced, with a resulting “waste” of the en- This equation conveys one of the most puzzling aspects of ergy required to deliver two electrons/protons, obtained from nitrogenase function, for it incorporates an obligatory formation hydrolysis of four ATP. We here report experiments that con- firm the essential mechanistic role for H formation, and hence of one mole of H2 per mole of N2 reduced, which requires the 2 waste of two reducing equivalents and four ATP (1, 2). A kinetic a limiting stoichiometry for biological nitrogen fixation of eight + → framework for nitrogenase function that incorporates the stoichi- electrons/protons, not six as in the equation N2 3H2 2NH3. ometry of Eq. 1 is provided by the Lowe–Thorneley model (1, 2, 4), These experiments were devised to test our recently proposed “ ” which describes transformations among catalytic intermediates reductive elimination mechanism for H2 formation and the activation of nitrogenase for ammonia production. Our find- (denoted En), where n is the number of electrons and protons ings provide direct experimental support for that mechanism. (n = 0–8) delivered to MoFe protein. N2 reduction requires activation of the MoFe protein to the E4 state in which FeMo-cofactor has accumulated four electrons and four protons stored as two Author contributions: B.M.H., D.R.D., and L.C.S. designed research; Z.-Y.Y., N.K., and L.C.S. – – performed research; Z.-Y.Y., N.K., D.L., B.M.H., D.R.D., and L.C.S. analyzed data; and hydrides that bridge Fe atoms [Fe H Fe] and two protons pre- Z.-Y.Y., D.L., B.M.H., D.R.D., and L.C.S. wrote the paper. fi – sumably bound to sul des of FeMo-cofactor (Fig. 1B)(5 8). The The authors declare no conflict of interest. binding of N to the E state is coupled to the stoichiometric 2 4 1To whom correspondence may be addressed. E-mail: [email protected], deandr@ evolution of one H2 per N2 reduced. vt.edu, or [email protected]. We recently presented a draft mechanism for N2 reduction by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nitrogenase that incorporates a mechanistic explanation for 1073/pnas.1315852110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1315852110 PNAS | October 8, 2013 | vol. 110 | no. 41 | 16327–16332 Downloaded by guest on September 27, 2021 A 6). In the absence of reactions with other substrates, this intermediate relaxes to the resting E0 level in a two-step process: (i) release of HD to form E2(D) followed by (ii) release of a second HD to form E0. The formation of the E4(2D) and E2(D) deuterated intermediates of Fig. 1 is unique to the re mechanism (5). In an attempt to intercept these intermediates, and thereby reveal their presence, turnover conditions were created that favor HD formation (0.25 atm N2 and 0.7 atm D2) (12), but in- B cluded a small amount of 13C-acetylene (typically 0.02 atm). As indicatedinFig.1andinDiscussion, reduction of C2H2 by E4(2D)ispredictedtoyieldC2H2D2 and possibly C2H3D, whereas reduction by E2(D) would yield C2H3D. These studies 13 used C2H2 to avoid confusion from the small but significant natural abundance of 13C (1.07%) (25) in acetylene. Most of the ethylene formed in either the presence or absence 13 C of N2 is C2H4, which is detected by mass spectrometry (MS) as a peak with mass/charge (m/z) of 30, following separation 13 from other gases by gas chromatography (GC). The C2H4 clearly forms by the normal acetylene reduction process, with the two protons added to C2H2 coming from solvent without intervention of D2 or N2. However, deuterated ethylenes were also formed 13 with the full C2H2/D2/N2 reaction mixture, and as expected from prior work, they were not formed when N2 was not included (or the reactions were conducted with H2 instead of D2), Fig. 2. 13 A substantial amount of monodeuterated ethylene ( C2H3D; m/z = 31) was detected (Fig. 1); its rate of formation was found Fig. 1. FeMo-cofactor and key mechanistic steps. (A) FeMo-cofactor in- to be ∼27 nmol C2H3D/nmol MoFe protein over 20 min (Fig. 2), organic core with Mo in magenta, Fe in rust, S in yellow, and C in gray. The 13 structure and numbering of Fe atoms are based on the protein database file equivalent to 2% of the amount of C2H4 generated. Most sig- fi 13 Protein Data Bank ID code 1M1N. The face composed of Fe atoms 2, 3, 6, and ni cantly, a product with m/z of 32, corresponding to C2H2D2 7 is noted with a red circle. (B) Proposed reductive elimination (re) mecha-

nism for N2 binding at E4 with re of H2 and hydrogenation of N2 to generate a metal bound diazene intermediate. Colors are N in blue and H in green. Fe6 0 in red indicates the Fe valence state. (C) Predicted steps for the N2-dependent formation of 2HD (right to left) during relaxation of E4(2D) formed by D2 displacement of N2. The possible interception of the key E4(2D) and E2(D) intermediates by acetylene to produce C2H3DandC2H2D2 is shown. In the absence of information about the structural rearrangements that are likely to occur during formation of intermediates, these visualizations are based on the resting FeMo-cofactor structure.

indicated in Fig. 1C and Fig. S1, the mechanism predicts that during turnover of enzyme in H2O buffer under D2/N2, the reverse reaction of the E4[N2/N2H2] intermediate with D2 would generate dideutero-E4 [E4(2D)], which can relax to monodeutero-E2 [E2(D)] through loss of HD (5, 22). Neither of these two deuterated enzymatic intermediates could otherwise form in H2O buffer, as D2 does not react with nitrogenase except through displacement of N2 (9, 11, 23). As indicated in Fig. 1C, the formation of E2(D) and E4(2D) should be revealed by intercepting them with the nonphysiological substrate acetylene (C2H2) (24) to generate deuterated ethylenes (C2H3DandC2H2D2). As reduction of C2H2 by nitrogenase in H2O buffer and in the 13 presence of D2 without added N2 would generate only C2H4, Fig. 2. Time-dependent formation of mono- and dideutero C-ethylene 13 without incorporation of D from D , the formation of these catalyzed by nitrogenase reduction of C2H2. Shown is the formation of 2 13 13 monodeutero- C-ethylene ( C2H3D) as a function of time determined by deuterated ethylenes during turnover under C2H2/D2/N2 would 13 GC/MS monitoring of m/z = 31 for a reaction mixture containing C2H2 and conﬁrm the essential mechanistic role for reversible H2 loss upon including D2 and N2 (■), just D2 (⊠), or H2 and N2 (□). The slight increase in N2 binding, and thus of the eight-electron stoichiometry for ni- m/z = 31 for the +D2 and the +H2/N2 controls can be attributed to in- ﬁ 1 13 trogen xation by nitrogenase embodied in Eq. , and provide ex- corporation of natural-abundance D into nominally C2H4 reduction prod- perimental support for the proposed re mechanism. uct; the difference between the two controls is a consequence of either or

both, reaction of the N2 substrate in the +H2/N2 control and/or a conse- Results quence of a trace amount of N2 contamination in the D2 control. (Inset) 13 13 = The earlier ﬁnding that HD is formed during turnover of nitro- Formation of dideutero- C-ethylene ( C2H2D2, m/z 32) monitored at m/ = 13 ● ◇ ○ – z 32 starting with C2H2/D2/N2 ( ), just D2 ( ), or H2/N2 ( ). The amounts genase under a mixture of D2/N2, but not under D2 alone (9, 12 13 of C2H2D2 are over 20 times higher than would arise from natural abun- 16, 23), can be explained by an re mechanism in which N2 is 13 dance D (∼0.0115%) incorporated in nominally C2H3D. The assays were displaced from the E [N /N H ] state by D through oxidative 13 4 2 2 2 2 conducted with partial pressures of 0.02 atm C2H2,0.25atmN2,and0.7atm addition to form the E4(2D) state containing two Fe-bridging H2/D2, where present. The molar ratio between Fe protein and MoFe protein deuterides [Fe–D–Fe] that are not exchangeable with solvent (5, was 2:1. All assays were incubated at 30 °C.

16328 | www.pnas.org/cgi/doi/10.1073/pnas.1315852110 Yang et al. Downloaded by guest on September 27, 2021 Fig. 3. Deuterated ethylene formation as a function of acetylene partial Fig. 4. Deuterated ethylene formation as a function of D2 partial pressure. pressure. Semilogarithmic plot of monodeuterated (C2H3D) or dideuterated Semilogarithmic plot of amounts of C2H3DandC2H2D2 formed versus the (C2H2D2) ethylene expressed as percentage of total ethylene formed, plotted partial pressure of D2. The partial pressure of C2H2 was 0.02 atm and the as a function of the partial pressure of acetylene. The partial pressure of D2 partial pressure of N2 was 0.2 atm. The molar ratio of Fe protein to MoFe was 0.7 atm and the partial pressure of N2 was 0.2 atm. The molar ratio of Fe protein was 4:1. Assay conditions as in Fig. 3. protein and MoFe protein was 4:1. Assay conditions as in Fig. 2, except all assays were incubated at 30 °C for 60 min.

reaction of C2H2 with those deuterated E states would yield C H D and C H D, respectively, as described in the previous was cleanly detected (Fig. 2, Inset), with a lower, although still 2 2 2 2 3 paragraph. Correspondingly, as shown in Fig. 4, the formation of signiﬁcant, production rate of ∼1.9 nmol 13C H D /nmol MoFe 2 2 2 both C H D and C H D increases in parallel with increasing D protein over 20 min. This species can only have been produced 2 3 2 2 2 2 13 partial pressure, as expected from this analysis. by C2H2 intercepting E4(2D) (Fig. 1), the intermediate formed The yield of C2H3D and C2H2D2 similarly increases in parallel through N2 replacement by D2. As indicated in Fig. 1 and Fig. S1, 13 13 with increasing partial pressure of N (Fig. 5). This can be 2 CHEMISTRY formation of C2H3D is likely to occur primarily through C2H2 explained by enhanced formation of E [N /N H ] by reaction of intercepting E2(D), but could also occur by an alternative channel 4 2 2 2 13 for the reaction of C2H2 with E4(2D). N2 with E4. Increased formation of E4[N2/N2H2] in turn would Reduction of acetylene and N2 are mutually exclusive, with enhance reaction with D2 to form E4(2D), which can be inter- complicated inhibition kinetics between these two substrates cepted by acetylene to form deuterated ethylenes (Fig. 1). (26). Therefore, it was of interest to determine the effect of varying the C2H2 partial pressure on the formation of C2H3D and C2H2D2 at a ﬁxed N2 and D2 concentration. As the partial

pressure of C2H2 is increased, the amounts of C2H3D and BIOCHEMISTRY C2H2D2 expressed as fractions of total ethylene formed decrease with increasing acetylene concentration, with the strongest decrease in the fraction of C2H3D (Fig. 3). The overall suppression of deuterated ethylene products can be understood as the result of increased capture by C2H2 of states less reduced than E4[N2/ N2H2], thereby preventing the formation of this state and its reaction with D2, which is required for the creation of deuterated ethylenes. It is also possible to explain the differential inﬂuence of increasing C2H2 concentration on the fraction of C2H2D2 and C2H3D being formed. Increasing the C2H2 concentration would enhance the tendency of any E4(2D) that does form to react with the C2H2 to form C2H2D2, somewhat relieving the suppression of C2H2D2 formation. This would further suppress C2H3D production, as enhanced reaction of E4(2D) would compete with HD release by E4(2D) to form the E2(D) state, whose reaction with C2H2 generates C2H3D. Davis et al. showed that when both N2 and C2H2 are included in a nitrogenase assay, increasing the partial pressure of H2 in- creasingly diverts nitrogenase from reduction of N2 to reduction of C2H2 (27), an observation interpreted as resulting from in- Fig. 5. Deuterated ethylene formation as a function of N2 partial pressure. creased reaction of H2 with E4[N2/N2H2], displacing N2 and Semilogarithmic plot of amounts of C H D and C H D formed as a function suppressing the formation of NH . The re mechanism further 2 3 2 2 2 3 of the partial pressure of N2. The partial pressure of C2H2 was 0.02 atm and predicts that in the presence of D2, the displacement of N2 the partial pressure of D2 was 0.6 atm. The molar ratio of Fe protein to MoFe generates E4(2D), which can release HD to form E2(D) (Fig. 1); protein was 4:1. Assay conditions as in Fig. 3.

Yang et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16329 Downloaded by guest on September 27, 2021 D2 can react with E4(N2/N2H2), replacing the N2 and undergoing oxidative addition to generate E4(2D). We recognized that this state in fact can be expected to react with C2H2 to form C2H2D2, with the idealized mechanism (Fig. S1) involving terminalization of an [Fe–D–Fe] bridge of E4 and migratory insertion of bound C2H2 into the Fe–D bond to form an Fe–alkenyl intermediate, followed by reductive elimination of C2H2D2 (17, 28). Previous studies (1, 4) could not distinguish reaction at the E4 state from reaction at the E2 state when C2H2 is reduced in the absence of N2,asN2 is required to enable gaseous D2 to enter the nitrogenase catalytic process. The possibility that acetylene can access different nitrogenase redox states, however, has been suggested on the basis of experiments using a nitrogenase variant that exhibits N2 reduction that is resistant to inhibition by acetylene (29). The E4(2D) state would also relax through the loss of HD to form E2(D), an E2 state whose unique isotopic composition can be generated in no other way. Interception of the E2(D) state by C2H2 would then generate C2H3D, with Fig. S1 presenting a plausible mechanism: hydride terminalization and insertion, followed by alkenyl protonolysis (17, 28); this reaction also might occur through an alternative reaction channel of E (2D), as fl 4 Fig. 6. Deuterated ethylene formation as a function of electron ux. noted in Fig. 1 and Fig. S1. Semilogarithmic plot of amounts of C2H3D and C2H2D2 formed as a function of the electron flux, as characterized by the ratio of Fe protein to MoFe Testing the Predictions. In this study, we have tested the pre- protein. The partial pressure of C2H2 was 0.02 atm, D2 was 0.68 atm, and N2 was 0.3 atm. The molar ratio of Fe protein to MoFe protein varied from 2:1 dictions of an involvement of gaseous D2 in substrate reduction to 32:1. Assay conditions as in Fig. 3. by using C2H2 reduction under N2/D2/C2H2 gas mixtures to intercept the E4(2D) and E2(D) states. As expected, the control reaction of turnover under D2/C2H2 generates only C2H4, with- fl The rate of electron ow through nitrogenase (called electron out incorporation of D from gaseous D2 to generate either fl ux) is known to affect product distribution for other substrates C2H3DorC2H2D2 (Fig. 2). In dramatic contrast, C2H2 reduction fl (1). The electron ux is easily controlled by altering the molar by nitrogenase under a N2/D2/C2H2 gas mixture in fact produces ratio of Fe protein to MoFe protein ([Fe protein]:[MoFe pro- readily measured amounts of C2H2D2 and even greater amounts tein]), the electron flux increasing with this ratio. In earlier of C2H3D (Fig. 2). The lower yield of C2H2D2 likely indicates studies, it was observed that varying the electron flux affects the that the probability for binding and reduction of C2H2 by E4(2D) HD formation rate (12). The current experiments show that as is substantially lower than that for the relaxation to E2(D) through the flux is increased in the presence of 0.02 atm C2H2, 0.68 atm loss of HD, and the reduction of C2H2 by E2(D) (Fig. 1). These D2, and 0.3 atm N2, the amounts of the two deuterated ethylene observations are enriched by the considerations of the dependence products decrease (Fig. 6). This observation suggests that the of the yields of C2H3D and C2H2D2 on the partial pressures of fl higher flux enhances electron delivery to the E4[N2/N2H2] in- C2H2,D2,N2, and electron ux, all of which are understandable termediate, sending it along the catalytic pathway to NH3 pro- in terms of the production of the E4(2D) and E2(D) states under duction and thus competitively decreasing the formation of the these turnover conditions, as predicted by the re mechanism for deuterated ethylenes. FeMo-cofactor activation for N2 binding and reduction. Note that the reduction of C2H2 to C2H3D by reaction with Discussion E2(D) formally corresponds to the reduction of C2H2 by the Predictions of the re Mechanism. Subsequent to formulation of the HD that otherwise would form during relaxation of E2(D) to E0, re mechanism for the activation of FeMo-cofactor to reduce N2 a perspective that highlights the contrast between this result, achieved in the presence of N , with the failure of nitrogenase to (Fig. 1) (5), we noted that addition of C2H2 to a N2/D2 reaction 2 mixture should offer a rigorous test of the mechanism. The test is use H2/D2 to reduce any substrate in the absence of N2. Elabo- founded on a defining characteristic of nitrogenase catalysis, an rating on this perspective, the formation of HD during turnover 2 exact distinction between hydrons (H/D/T) associated with the under N2/D2, with stoichiometry (Eq. ) (1) gaseous diatomics, H2/D2/T2, and those derived from solvent + − D + 2H ðaqÞ + 2e N2 ! 2HD; [2] water. Thus, when nitrogenase in protic buffer is turned over 2 Nitrogenase under N2/D2, gaseous D2 can displace N2 from the E4(N2/N2H2) state (Fig. 1), stoichiometrically yielding two HD (11). This and can be seen to correspond to the nitrogenase-catalyzed reduction other observations clearly show that diatomic H2/D2 is not used of protons by D2 and electrons with N2 as cocatalyst, as the re- to reduce N2 during turnover under N2//H2/D2/T2 (in particular, action does not proceed without N2. Likewise, although C2H2D2 T incorporated into the ammonia product of N2 fixation would is well known to form during nitrogenase reduction of C2D2 in exchange with solvent) (1). Likewise, as demonstrated here (Fig. H2Obuffer(orC2H2 in D2O buffer) (30), formation of this 2), when C2H2 is reduced in the presence of D2, no deuterated species during turnover under N2/D2/C2H2 corresponds to the ethylenes are generated. previously unobserved reduction of C2H2 by gaseous D2 with 3 With this foundation, we recognized that the re mechanism N2 as cocatalyst (Eq. ): predicts that turnover under C2H2/D2/N2 should not only in- D + C H N2 ! C H D : [3] corporate H from solvent to generate C2H4 by the normal re- 2 2 2 Nitrogenase 2 2 2 duction process, but through the agency of the added N2 also should breach the separation of gaseous D2 from solvent protons Correspondingly, the formation of C2H3D involves incorporation − + by generating both C2H3D and C2H2D2. According to the re of D derived from D2 along with H from solvent with N2 mechanism (Fig. 1), when turnover is carried out under N2/D2, as cocatalyst.

16330 | www.pnas.org/cgi/doi/10.1073/pnas.1315852110 Yang et al. Downloaded by guest on September 27, 2021 Conclusions 10 mM ATP, 60 mM phosphocreatine, 1.2 mg/mL BSA, and 0.4 mg/mL cre- atine phosphokinase) in a 100-mM Mops [3-(N-morpholino)propanesulfonic i) The incorporation of D from D2 into the nitrogenase reduc- acid] buffer, pH 7.0. Before all assays, the MoFe protein was added and the vials were equilibrated for about 20 min. The gas overpressure was released tion products C2H2D2 and C2H3D during turnover under before assays were begun. Reactions were initiated by the addition of Fe C2H2/D2/N2 in H2O demonstrates the presence of the protein and the reaction was allowed to proceed at 30 °C for 60 min, unless E4(2D) and E2(D) states under these conditions. noted otherwise. Reactions were quenched by the addition of 300 μLof ii) This incorporation provides a very clear demonstration of the 400 mM EDTA, pH = 8.0, solution. For the acetylene partial pressure de- essential mechanistic role for obligatory, reversible loss of H2 pendence study, 3.3 mL of the assay buffer was used with acetylene partial upon N2 binding and thus of the eight-electron stoichiometry pressures from 0.002 to 0.008 atm with the same protein concentrations as for nitrogen fixation by nitrogenase embodied in Eq. 1. Until described below. For analysis of the partial pressure dependence of acetylene, N , and D , all samples contained 1.0 mg of wild-type MoFe protein now, the data indicating that some H2 must be evolved during 2 2 and 1.0 mg of Fe protein in 1.1 mL of assay buffer. For the electron flux N2 reduction have been viewed as being much more compelling than the data indicating an obligatory evolution of one dependence study, 1.0 mg of MoFe protein was used and the Fe protein varied from 0.5 to 8.0 mg in 1.1 mL of assay buffer. In the time course study H2 for every N2 reduced, leading to the stoichiometry of 13 1 using C2H2, 1.0 mg of wild-type MoFe protein and 0.5 mg of Fe protein in Eq. (1). 1.1 mL of assay buffer. The acetylene partial pressure dependence study was done in triplicate was used. Experiments to monitor the time dependence iii) The formation of E4(2D) and E2(D) during turnover under of formation of deuterated ethylenes were done over 20 min, before limi- D2/N2 in H2O is predicted by the re mechanism for the acti- tation by a component of the assay, whereas experiments to maximize vation of FeMo-cofactor for reduction of N2 (Fig. 1), and the product formation were conducted over 60 min. interception of these intermediates by C2H2 thus provides direct experimental evidence in support of this mechanism. GC-MS Measurement of Products. Ethylene was quantified by analysis of the iv) The well-known reduction of protons by D2 to form 2HD headspace gas with a Shimadzu GC-2010 gas chromatograph equipped with a programmed temperature vaporization (PTV) injector and a Shimadzu during turnover under D2/N2 in H2O and the reductions of GCMS-QP2010S mass spectrometer in selected ion monitoring mode using C2H2 by D2/N2 reported here should be viewed as being electron ionization as the ion source. Separation of ethylene from other catalyzed by nitrogenase with N2 as cocatalyst. gases was achieved with an Rt-Alumina BOND/KCl column (30 m, 0.32-mm i.d., and 5.0-μm film thickness) (Restek). The injector and column temper- Materials and Methods atures were set to 35 °C. Ultrapure helium was used as the carrier gas set at Chemical Reagents and Protein Purification. Gases were purchased from Air a linear velocity of 60 cm/s. For each injection, 100 or 150 μL of headspace 12 Liquide. Ultrapure helium was purchased from Airgas. Ethylene (99.9% vol/ gas was directly injected into the PTV injector. When C-acetylene (C2H2) 13 13 vol) was obtained from Praxair Inc. C-acetylene (99 atom % C) and D2 (99.8 was the substrate, ethylene (C2H4, mass 28) was monitored at m/z = 28 (m), atom % D) were purchased from Sigma-Aldrich and Isotec, respectively. All with minor species being 29 (m+1) and 30 (m+2) resulting from natural fi 2 13 13 other reagents were obtained from Sigma-Aldrich or Fisher Scienti c and abundance H (D) and C in the acetylene. For samples using C2H2, fi fi 13 were used without further puri cation, unless speci ed otherwise. Azoto- ethylene ( C2H4, mass 30) was monitored as m/z = 30 (m) with minor bacter vinelandii strains DJ1260 (hydrogenase genes removed) and DJ884 products detected at 31 (m+1) and 32 (m+2) from natural abundance 2H.

(R187I) were grown as previously described. The wild-type nitrogenase MoFe When the reaction was run with D2 in place of H2, the ethylene products CHEMISTRY protein (DJ1260) and Fe protein (DJ884) were expressed and purified 13 13 C2DH3 (mass 29) and C2D2H2 (mass 30) or C2DH3 (mass 31) and C2D2H2 as previously reported (31, 32). The wild-type MoFe protein in this study (mass 32) were detected. Ethylene was quantified from the peak area using contains a seven-His tag addition near the carboxyl-terminal end of the a standard curve generated with known amounts of ethylene in argon. α -subunit. Protein concentrations were determined by the Biuret assay using It was taken that all isotopomers of ethylene had the same retention time > BSA as standard. The purities of these proteins were estimated to 95% and that the ionization and fragmentation efficiency of all isotopomers was based on denaturing polyacrylamide gel separation with Coomassie blue the same. Intensities for all ethylene peaks were established by subtraction of staining. Manipulation of proteins, solutions, and buffers was done in sep- the signal background at that retention time. tum-sealed serum vials under an argon atmosphere on a Schlenk vacuum line. All gases and liquid transfers used gas-tight syringes. ACKNOWLEDGMENTS. We thank Dr. Bradley D. Wahlen for his kind help BIOCHEMISTRY with GC-MS experiments. We thank Profs. Robert Hausinger, Patrick Assay Methods. All assays were conducted in 9.4-mL serum vials with gases Holland, and Jonas Peters for insightful comments. This work was supported added before addition of 1.1 mL of an anaerobic “assay buffer” consisting of by the National Institutes of Health GM59087 (to L.C.S. and D.R.D.) and 90 mM sodium dithionite, a MgATP regenerating system (13.4 mM MgCl2, HL 13531 (to B.M.H.).

1. Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96(7): 10. Wherland S, Burgess BK, Stiefel EI, Newton WE (1981) Nitrogenase reactivity: Effects 2983–3012. of component ratio on electron flow and distribution during nitrogen fixation. Bio- 2. Thorneley RNF, Lowe DJ (1985) Kinetics and Mechanism of the Nitrogenase Enzyme chemistry 20(18):5132–5140. System. Molybdenum Enzymes, Metal Ions in Biology, ed Spiro TG (Wiley-Interscience, 11. Burgess BK, Wherland S, Newton WE, Stiefel EI (1981) Nitrogenase reactivity: Insight – New York), Vol 7, pp 221 284. into the nitrogen-fixing process through hydrogen-inhibition and HD-forming re- 3. Simpson FB, Burris RH (1984) A nitrogen pressure of 50 atmospheres does not prevent actions. Biochemistry 20(18):5140–5146. evolution of hydrogen by nitrogenase. Science 224(4653):1095–1097. 12. Guth JH, Burris RH (1983) Inhibition of nitrogenase-catalyzed NH3 formation by H2. 4. Wilson PE, Nyborg AC, Watt GD (2001) Duplication and extension of the Thorneley Biochemistry 22(22):5111–5122. and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using 13. Li JL, Burris RH (1983) Influence of pN2 and pD2 on HD formation by various nitro- a MATHEMATICA software platform. Biophys Chem 91(3):281–304. genases. Biochemistry 22(19):4472–4480. 5. Hoffman BM, Lukoyanov D, Dean DR, Seefeldt LC (2013) Nitrogenase: A draft 14. Jensen BB, Burris RH (1985) Effect of high pN2 and high pD2 on NH3 production, H2 mechanism. Acc Chem Res 46(2):587–595. evolution, and HD formation by nitrogenases. Biochemistry 24(5):1141–1147. 6. Igarashi RY, et al. (2005) Trapping H- bound to the nitrogenase FeMo-cofactor active 15. Dilworth MJ, Fisher K, Kim CH, Newton WE (1998) Effects on substrate reduction of site during H2 evolution: Characterization by ENDOR spectroscopy. J Am Chem Soc substitution of histidine-195 by glutamine in the alpha-subunit of the MoFe protein 127(17):6231–6241. – 7. Lukoyanov D, Yang Z-Y, Dean DR, Seefeldt LC, Hoffman BM (2010) Is Mo involved in of Azotobacter vinelandii nitrogenase. Biochemistry 37(50):17495 17505. 16. Fisher K, Dilworth MJ, Newton WE (2000) Differential effects on N(2) binding and hydride binding by the four-electron reduced (E4) intermediate of the nitrogenase MoFe protein? J Am Chem Soc 132(8):2526–2527. reduction, HD formation, and azide reduction with alpha-195(His)- and alpha-191 8. Doan PE, et al. (2011) 57Fe ENDOR spectroscopy and ‘electron inventory’ analysis of (Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase. Biochemistry – the nitrogenase E4 intermediate suggest the metal-ion core of FeMo-cofactor cycles 39(50):15570 15577. through only one redox couple. J Am Chem Soc 133(43):17329–17340. 17. Crabtree RH (2009) The Organometallic Chemistry of the Transition Metals (Wiley, 9. Jackson EK, Parshall GW, Hardy RW (1968) Hydrogen reactions of nitrogenase. For- Hoboken, NJ), 5th Ed, p xiii. mation of the molecule HD by nitrogenase and by an inorganic model. J Biol Chem 18. Ballmann J, Munhá RF, Fryzuk MD (2010) The hydride route to the preparation of 243(19):4952–4958. dinitrogen complexes. Chem Commun (Camb) 46(7):1013–1025.

Yang et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16331 Downloaded by guest on September 27, 2021 19. Kubas GJ (2007) Fundamentals of H2 binding and reactivity on transition metals un- 26. Seefeldt LC, Dance IG, Dean DR (2004) Substrate interactions with nitrogenase: Fe derlying hydrogenase function and H2 production and storage. Chem Rev 107(10): versus Mo. Biochemistry 43(6):1401–1409. 4152–4205. 27. Davis LC, Shah VK, Brill WJ (1975) Nitrogenase. VII. Effect of component ratio, ATP 20. Peruzzini M, Poli R, eds (2001) Recent Advances in Hydride Chemistry (Elsevier Science, and H2 on the distribution of electrons to alternative substrates. Biochim Biophys Acta Amsterdam). 403(1):67–78. 21. Lowe DJ, Thorneley RN (1984) The mechanism of Klebsiella pneumoniae nitrogenase 28. Hartwig J (2010) Organotransition Metal Chemistry: From Bonding to Catalysis action. The determination of rate constants required for the simulation of the kinetics (University Science Books, Sausalito, CA). of N reduction and H evolution. Biochem J 224(3):895–901. 2 2 29. Christiansen J, Cash VL, Seefeldt LC, Dean DR (2000) Isolation and characterization of 22. Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM (2007) Connecting ni- an acetylene-resistant nitrogenase. J Biol Chem 275(15):11459–11464. trogenase intermediates with the kinetic scheme for N reduction by a relaxation 2 30. Seefeldt LC, Yang ZY, Duval S, Dean DR (2013) Nitrogenase reduction of carbon- protocol and identification of the N2 binding state. Proc Natl Acad Sci USA 104(5): containing compounds. Biochim Biophys Acta 1827(8-9):1102–1111. 1451–1455. 31. Christiansen J, Goodwin PJ, Lanzilotta WN, Seefeldt LC, Dean DR (1998) Catalytic and 23. Hoch GE, Schneider KC, Burris RH (1960) Hydrogen evolution and exchange, and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB- conversion of N2OtoN2 by soybean root nodules. Biochim Biophys Acta 37:273–279. 24. Dilworth MJ (1966) Acetylene reduction by nitrogen-fixing preparations from Clos- deletion mutant of Azotobacter vinelandii. Biochemistry 37(36):12611–12623. tridium pasteurianum. Biochim Biophys Acta 127(2):285–294. 32. Seefeldt LC, Mortenson LE (1993) Increasing nitrogenase catalytic efficiency for + 25. Böhlke JK, et al. (2005) Isotopic compositions of the elements. 2001. J Phys Chem Ref MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn2 to show + Data 34(1):57–67. interaction of serine 16 with Mg2 . Protein Sci 2(1):93–102.

16332 | www.pnas.org/cgi/doi/10.1073/pnas.1315852110 Yang et al. Downloaded by guest on September 27, 2021