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

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.

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Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.