Dmitriy Lukoyanov

Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage

Brian M. Hoffman,* Dmitriy Lukoyanov, Zhi-Yong Yang,† Dennis R. Dean,*,‡ and Lance C. Seefeldt*,† †Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322, United States ‡Department of Biochemistry, Virginia Tech, 900 West Campus Drive, Blacksburg, Virginia 24061, United States Departments of Chemistry and Molecular Biosciences, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States

Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state.

A major obstacle to understanding the reduction of N2 to NH3 by nitrogenase has been the impossibility of synchronizing electron delivery to the MoFe protein for generation of specific enzymatic intermediates. When an intermediate is trapped without synchronous electron delivery, the number of electrons, n, it has accumulated is unknown. Consequently, the intermediate is untethered from kinetic schemes for reduction, which are indexed by n. We show that a trapped intermediate itself provides a “synchronously prepared” initial state, and its relaxation to the resting state at 253 K, conditions that prevent electron delivery to MoFe protein, can be analyzed to reveal n and the nature of the relaxation reactions. The approach is applied to the “H+/H− intermediate” (A) that appears during turnover both in the presence and absence of N2 substrate. A exhibits an S = ½ EPR signal from the active-site iron–molybdenum cofactor (FeMo-co) to which are bound at least two hydrides/protons. A undergoes two-step relaxation to the resting state (C): A → B → C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE ≈ 3–4 (85% D2O). In the context of the Lowe–Thorneley kinetic scheme for N2 reduction, these results provide powerful evidence that H2 is formed in both relaxation steps, that A is the catalytically central state that is activated for N2 binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons.

A methyldiazene (HN N CH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

Methyldiazene (HNNCH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or 2H were used as substrates for the nitrogenase α-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = ½ state that has been characterized by EPR and 1H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt–Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before N-N bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to N-N bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before N-N cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in α-195Gln-substituted MoFe protein.

Is Mo Involved in Hydride Binding by the Four-Electron Reduced (E4) Intermediate of the Nitrogenase MoFe Protein?

We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the alpha-70(Ile) MoFe protein is freeze-trapped during H(+) reduction under Ar, the majority of the resting state EPR signal from the molybdenum-iron cofactor (FeMo-co) disappears and is replaced by the S = 1/2 signal of an intermediate that has been shown to be the E(4) MoFe state, which is activated for N(2) binding and reduction through the accumulation of 4 electrons/protons by FeMo-co. ENDOR studies of E(4) showed that it contains two hydrides bound to FeMo-co. We calculate that Mo involvement in hydride binding would require a vector-coupling coefficient for Mo of |K(Mo)| approximately > 0.2 and determine K(Mo) for the E(4) intermediate state through 35 GHz ENDOR measurements of a (95)Mo enriched MoFe protein, further comparing the results with those for the E(0) resting state. The experiments show that Mo of the resting-state FeMo-co is perturbed by the alpha-70(Ile) substitution and that the isotropic (95)Mo hyperfine coupling in E(4) is a(iso) approximately 4 MHz, less than that for the resting state. The decrease in a(iso) for (95)Mo of E(4) from the already small value in the resting state MoFe protein strongly suggests that the resting Mo(IV) is not one-electron reduced during the accumulation of the four electrons of E(4). In any case, the effective K for Mo is very small; |K(Mo)| approximately < 0.04, at least 5-fold less than the lower bound required for Mo to be involved in forming a Mo-H-Fe, hydride. As the hydride couplings also are both far too small and of the wrong symmetry to be associated with a terminal hydride on Mo, we may thus conclude that Mo does not participate in binding a hydride of the catalytically central E(4) intermediate and that only Fe ions are involved. Nonetheless, the response of the Mo coupling to subtle conformational changes in E(0) and to the formation of E(4) suggests that Mo is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states.

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

Significance 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 of one H2 per N2 reduced, with a resulting “waste” of the energy required to deliver two electrons/protons, obtained from hydrolysis of four ATP. We here report experiments that confirm the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, not six as in the equation N2 + 3H2 → 2NH3. These experiments were devised to test our recently proposed “reductive elimination” mechanism for H2 formation and the activation of nitrogenase for ammonia production. Our findings provide direct experimental support for that mechanism. Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 bound to FeMo-cofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H+ with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.

Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N2 for H2

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

Trapping an Intermediate of Dinitrogen (N2) Reduction on Nitrogenase

Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.

Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase

Nitrogenase catalyzes the reduction of N2 and protons to yield two NH3 and one H2. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH3), or hydrazine as substrate recently was shown to trap a common intermediate, designated I. It was further concluded that the two N-atoms of N2 are hydrogenated alternately (“Alternating” (A) pathway). In the present work, Q-band CW EPR and 95Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of 14,15N isotopologs plus 1,2H isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N2 reduction by nitrogenase.

Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.

A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates.

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe–H–Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1–8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped “hydride” state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.