Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 × 10(7) per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.

The reduction of N2 to NH3 is a requisite transformation for life.1 While it is widely appreciated that the iron-rich cofactors of nitrogenase enzymes facilitate this transformation,2-5 how they do so remains poorly understood. A central element of debate has been the exact site(s) of nitrogen coordination and reduction.6,7 The synthetic inorganic community placed an early emphasis on Mo8, because Mo was thought to be an essential element of nitrogenases3 and because pioneering work by Chatt and coworkers established that well-defined Mo model complexes could mediate the stoichiometric conversion of N2 to NH3.9 This chemical transformation can be performed in a catalytic fashion by two well-defined molecular systems that feature Mo centres.10,11 However, it is now thought that Fe is the only transition metal essential to all nitrogenases,3 and recent biochemical and spectroscopic data has implicated Fe instead of Mo as the site of N2 binding in the FeMo-cofactor.12 In this work, we describe a tris(phosphine)borane-supported Fe complex that catalyzes the reduction of N2 to NH3 under mild conditions, wherein >40% of the H+/e- equivalents are delivered to N2. Our results indicate that a single Fe site may be capable of stabilizing the various NxHy intermediates generated en route to catalytic NH3 formation. Geometric tunability at Fe imparted by a flexible Fe-B interaction in our model system appears to be important for efficient catalysis.13-15 We propose that the interstitial light C-atom recently assigned in the nitrogenase cofactor may play a similar role,16,17 perhaps by enabling a single Fe site to mediate the enzymatic catalysis via a flexible Fe-C interaction.18

Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pKa of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious autooxidation of tyrosine and tryptophan residues in the enzyme’s framework. Here we report the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥ 90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and X-ray absorption spectroscopies, we determine a pKa value for this compound of 11.9. Marcus theory analysis indicates that this elevated pKa results in a >10,000 fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

Biological N2 fixation to NH3 may proceed at one or more Fe sites in the active-site cofactors of nitrogenases. Modeling individual e−/H+ transfer steps of iron-ligated N2 in well-defined synthetic systems is hence of much interest but remains a significant challenge. While molecular Fe species have been recently demonstrated to catalyze the formation of NH3 from N2, mechanistic details of these processes remain elusive. Herein, we report the synthesis and isolation of a diamagnetic, 5-coordinate formally iron(IV) Fe═NNH2+ species supported by a tris(phosphino)silyl ligand via the direct protonation of a terminally bound Fe-N2− complex. The Fe═NNH2+ complex is redox-active, and low-temperature spectroscopic data and DFT calculations evidence an accumulation of significant radical character on the hydrazido ligand upon one-electron reduction to S = 1/2 Fe═ NNH2. At warmer temperatures, Fe═NNH2 rapidly converts to an iron hydrazine complex, Fe-NH2NH2+, via the additional transfer of proton and electron equivalents in solution. Fe-NH2NH2+ can liberate ammonia, and the sequence of reactions described here demonstrates that an iron site can shuttle from a distal intermediate (Fe═NNH2+) to an alternating intermediate (Fe-NH2NH2+) en route to NH3 liberation from N2. It is interesting to consider the possibility that similar “hybrid” mechanisms for N2 reduction may be operative in biological N2 fixation.

The ability of certain transition metals to mediate the reduction of N2 to NH3 has attracted broad interest in the biological and inorganic chemistry communities. Early transition metals such as Mo and W readily bind N2 and mediate its protonation at one or more N atoms to furnish M(NxHy) species that can be characterized and, in turn, extrude NH3. By contrast, the direct protonation of Fe-N2 species to Fe(NxHy) products that can be characterized has been elusive. Herein we show that addition of acid at low temperature to [(TPB)Fe(N2)][Na(12-crown-4)] results in a new S = 1/2 Fe species. EPR, ENDOR, Mössbauer, and EXAFS analysis, coupled with a DFT study, unequivocally assign this new species as [(TPB)Fe≡N-NH2]+, a doubly protonated hydrazido(2-) complex featuring an Fe-to-N triple bond. This unstable species offers strong evidence that the first steps in Fe-mediated nitrogen reduction by [(TPB)Fe(N2)][Na(12-crown-4)] can proceed along a distal or `Chatt-type' pathway. A brief discussion of whether subsequent catalytic steps may involve early or late stage cleavage of the N-N bond, as would be found in limiting distal or alternating mechanisms, respectively, is also provided.