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

A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems.

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Nitrogen is fundamental to all of life and many industrial processes. The interchange of nitrogen oxidation states in the industrial production of ammonia, nitric acid, and other commodity chemicals is largely powered by fossil fuels. A key goal of contemporary research in the field of nitrogen chemistry is to minimize the use of fossil fuels by developing more efficient heterogeneous, homogeneous, photo-, and electrocatalytic processes or by adapting the enzymatic processes underlying the natural nitrogen cycle. These approaches, as well as the challenges involved, are discussed in this Review.

Nitrogen-fixing bacteria catalyze the reduction of dinitrogen (N2) to two ammonia molecules (NH3), the major contribution of fixed nitrogen into the biogeochemical nitrogen cycle. The most widely studied nitrogenase is the Mo-dependent enzyme. The reduction of N2 by this enzyme involves the transient interaction of two component proteins, designated the Fe protein and the MoFe protein, and minimally requires sixteen MgATP, eight protons, and eight electrons. The current state of knowledge on how these proteins and small molecules together effect the reduction of N2 to ammonia is reviewed. Included is a summary of the roles of the Fe protein and MgATP hydrolysis, information on the roles of the two metal clusters contained in the MoFe protein in catalysis, insights gained from recent success in trapping substrates and inhibitors at the active site metal cluster FeMo-cofactor, and finally, considerations of the mechanism of N2 reduction catalyzed by nitrogenase.

The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

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...

Conspectus “Nitrogen fixation”—the reduction of dinitrogen (N2) to two ammonia (NH3) molecules—by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase make it the ‘Everest of enzymes’. This Account describes our efforts to climb one “face” of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMocofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, we need to discriminate between the two classes of potential reaction pathways: 1) a “distal” (D) pathway, in which H atoms add sequentially at a single N or 2) an “alternating” (A) pathway, in which H atoms add alternately to the two N atoms of N2. Secondly, we need to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires us to experimentally determine both the number of electrons/protons delivered to the MoFe protein and their “inventory”—a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this “climb” has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-cofactor as the specific site of reactivity, indicated that the sidechain of residue α70V controls access to this face, and supported the involvement of the sidechain of residue α195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use ENDOR/ESEEM spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this “face” of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.