Introduction 4041 2. Background 4043 2.1. Kinetics and Stoichiometry 4043 2.2. Trapping and Characterization of Substrates 4044 3. Intermediates of Nitrogenase Activation 4044 3.1. E 1 −E 3 4044 3.2. E 4 : The "Janus Intermediate" 4044 3.3. Redox Behavior and Hydride Chemistry of E 1 −E 3 : Why Such a Big Catalytic Cluster? 4046 3.4. Why Does Nitrogenase Not React with H 2 / D 2 /T 2 in the Absence of N 2 ? 4047 4. "Dueling" N 2 Reduction Pathways 4047 5. Intermediates of N 2 Reduction: E n , n ≥ 4 4048 5.1. Intermediate I 4048 5.2. Nitrogenase Reaction Pathway: D versus A 4048 5.3. Intermediate H 4049 6. Unification of the Nitrogenase Reaction Pathway with the LT Kinetic Scheme 4050 7. Obligatory Evolution of H 2 in Nitrogen Fixation: Reductive Elimination of H 2 4050 7.1. Hydride Protonation (hp) Mechanism 4051 7.2. Reductive Elimination (re) Mechanism 4051 7.3. Mechanistic Constraints Reveal That Nitrogenase Follows the re Mechanisms 4051 8. Test of the re Mechanism 4052 8.1. Predictions 4053 8.2. Testing the Predictions 4053 9. Completing the Mechanism of Nitrogen Fixation 4054 9.1. Uniqueness of N 2 and Nitrogenase 4055 9.2. Structure of the E 4 (N 2 ) Intermediate: Some Implications 4056 10. Summary of Mechanistic Insights 4056 10.1. Catalytic Intermediates of N 2 Fixation 4056 10.2. re Mechanism 4056 10.3. Turnover under N 2 /D 2 /C 2 H 2 as a Test of the re Mechanism 4057 11. Conclusions 4057 Associated Content 4057 Supporting Information 4057 Author Information 4057 Corresponding Authors 4057 Notes 4058 Biographies 4058 Acknowledgments 4058 References 4058

Conspectus Biological nitrogen fixation — the reduction of N2 to two NH3 molecules — supports more than half the human population. This reaction is catalyzed by the enzyme nitrogenase, whose predominant form, discussed here, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Nitrogenase has been studied extensively but the catalytic mechanism has remained unknown. At minimum, a mechanism must identify and characterize each intermediate formed during catalysis, and embed these intermediates within a kinetic framework that explains their dynamic interconversion. Nitrogenase kinetics have been described by the Lowe-Thorneley (LT) model, which provides rate constants for transformations among intermediates, denoted En, indexed by the number of electrons (and protons), n, that have been accumulated within the MoFe protein. However, until recently, research on purified nitrogenase had not resulted in characterization of any En state beyond Eo. In this article we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis, and their placement within the LT kinetic scheme. First we discuss the key E4 state, which is primed for N2 binding and reduction and which we refer to as the “Janus intermediate”. This state contains the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C-homocitrate]; FeMo-co) at its resting oxidation level, its four accumulated reducing equivalents being stored as two [Fe-H-Fe] bridging hydrides. The other two trapped intermediates contain reduced forms of N2. One, intermediate I, has S = 1/2 FeMo-co. ENDOR/HYSCORE measurements indicate that I, is the final catalytic state, E8, having NH3 product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (Non-Kramers; S ≥ 2). ESEEM measurements indicate that H binds the [−NH2] fragment and therefore corresponds to E7. These assignments, plus consideration of previous studies, imply a pathway in which (i) N2 binds at E4 with liberation of H2, (ii) N2 is promptly reduced to N2H2, (iii) the two N’s are hydrogenated alternately to form hydrazine-bound FeMo-co, and (iv) two NH3 are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a ‘Prompt-Alternating (P-A)’ reaction pathway, and unifies the catalytic pathway with the LT kinetic framework. However, it does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H2 upon N2 binding that apparently ‘wastes’ two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. The finding that E4 stores its four accumulated reducing equivalents as two bridging hydrides, considered in the context of the organometallic chemistries of hydrides and dihydrogen, leads us to propose an answer to this puzzle. Namely, that H2 release upon N2 binding involves reductive elimination of two hydrides to yield N2 bound to doubly reduced Fe. Coupled delivery of the two ava...

Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to support food and chemical production and the heavy reliance of the industrial Haber–Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.

We have 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-70Val→Ile substituted 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-cofactor. In the present work, EPR/ENDOR and photophysical measurements show that a state freeze-trapped during N2 reduction by wild type (WT) MoFe protein is the same Janus intermediate, thereby establishing the α-70Val→Ile intermediate as a reliable guide to mechanism, and enabling new experimental tests of the re/oa mechanism with WT enzyme. These allow us to show that the re/oa mechanism accounts for the longstanding Key Constraints on mechanism. Monitoring the S = ½ FeMo-co EPR signal of Janus 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 explaining earlier observations and verifying the re/oa mechanism. 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. The relative populations of E4(2N2H) and E4(4H) freeze-trapped during WT turnover furthermore show that the rapidly reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is roughly thermoneutral (ΔreG0 ~ −2 kcal/mol), whereas hydrogenation of gas-phase N2 would be highly endergonic. These findings establish (i) that re/oa satisfies all key constraints on mechanism, (ii) that Janus is the key 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 the nitrogenase MoFe protein carries out one of the most challenging chemical transformation in biology, the reduction of the N≡N triple bond.

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 N 2 substrate. A exhibits an S ‫؍‬ 1 ⁄2 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 3 B 3 C, where B has an S ‫؍‬ 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE Ϸ 3-4 (85% D 2 O). In the context of the Lowe-Thorneley kinetic scheme for N 2 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 N 2 binding by the accumulation of n ‫؍‬ 4 electrons, and that B has accumulated n ‫؍‬ 2 electrons.intermediate ͉ kinetic isotope effect

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 (re) 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.

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) (“Janus”) state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (k HP) but significant differences in the rate constant for H2 re with N2 binding/reduction (k re ). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (k HP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (k re ) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9:homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 35 GHz electron-nuclear double resonance (ENDOR) measurements on 14/15N and 12/13C isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X not equal N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either 14/15N or 13C: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A(14/15NX) < 0.07/0.1 MHz, A(13CX) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = 14/15N/13C/17O, nuclear quadrupole coupling constant e2qQ for X = 14N, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjunction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7" spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g2 are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if aiso(14/15NX) < 0.03-0.07/0.05-0.1 MHz and aiso(13CX) < 0.05-0.1 MHz, compared with calculated values of aiso(14/15NX) = 0.3/0.4 MHz and aiso(13CX) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.