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

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.

We have employed gamma-irradiation at cryogenic temperatures (77 K and also approximately 6 K) of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam to inject the "second" electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressively higher temperatures. (i) The primary product upon reduction of oxyP450 4 is the end-on, "H-bonded peroxo" intermediate 5A. (ii) This converts even at cryogenic temperatures to the hydroperoxo-ferriheme species, 5B, in a step that is sensitive to these mutations. Yields of 5B are as high as 40%. (iii) In WT and D251N P450s, brief annealing in a narrow temperature range around 200 K causes 5B to convert to a product state, 7A, in which the product 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but less extensive) results are seen for the alternate substrate, adamantane. (iv) Although the T252A mutation does not interfere with the formation of 5B, the cryoreduced oxyT252A does not yield product, which suggests that 5B is a key intermediate at or near the branch-point that leads either to product formation or to nonproductive "uncoupling" and H(2)O(2) production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) There is no spectroscopic evidence for the buildup of a high-valence oxyferryl/porphyrin pi-cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H(2)O and D(2)O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe(3+), and that the proton removed from the C(5) carbon of substrate during hydroxylation is trapped as the hydroxyl proton. This demonstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of 6. (vi) Annealing at > or = 220 K converts the initial product state 7A to the equilibrium product state 7, with the transition occurring via a second nonequilibrium product state, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxyl proton no longer is trapped. (vii) The present results are discussed in the context of other efforts to detect intermediates in the P450 catalytic cycle.