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.

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.

Nitrogenase MoFe protein immobilization yields a bioelectrode capable of producing H2 and NH3 independent of the ATP-hydrolyzing Fe protein.

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.

We recently demonstrated that N2 reduction by nitrogenase involves the obligatory release of one H2 per N2 reduced. These studies focused on the E4(4H) ‘Janus intermediate’, which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. E4(4H) is poised to bind and reduce N2 through reductive elimination (re) of the two hydrides as H2, coupled to the binding/reduction of N2. To obtain atomic-level details of the re activation process, we carried out in situ 450 nm photolysis of E4(4H) in an EPR cavity at temperatures below 20 K. ENDOR and EPR measurements show that photolysis generates a new FeMo-co state, denoted E4(2H)*, through the photoinduced re of the two bridging hydrides of E4(4H) as H2. During cryoannealing at temperatures above 175 K, E4(2H)* reverts to E4(4H) through the oxidative addition (oa) of the H2. The photolysis quantum yield is temperature invariant at liquid helium temperatures and shows a rather large kinetic isotope effect, KIE = 10. These observations imply that photoinduced release of H2 involves a barrier to the combination of the two nascent H atoms, in contrast to a barrierless process for mono-metallic inorganic complexes, and further suggest that H2 formation involves nuclear tunneling through that barrier. The oa recombination of E4(2H)* with the liberated H2 offers compelling evidence for the Janus intermediate as the point at which H2 is necessarily lost during N2 reduction; this mechanistically coupled loss must be gated by N2 addition that drives the re/oa equilibrium toward reductive elimination of H2 with N2 binding/reduction.

Nitrogenase is activated for N 2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E 4 , which contains two iron-bridging hydrides [Fe-H-Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H 2 per N 2 reduced, which would "waste" two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H 2 production. In this model, H 2 is produced by reductive elimination of the two bridging hydrides of E 4 during N 2 binding. This process releases H 2 , yielding N 2 bound to FeMocofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D 2 /N 2 , the reverse reaction of D 2 with the N 2 -bound product of reductive elimination would generate dideutero-E 4 [E 4 to two ammonia (NH 3 ) molecules-is primarily catalyzed by the Mo-dependent nitrogenase. This enzyme comprises an electron-delivery Fe protein and MoFe protein, which contains the active site FeMo-cofactor ( Fig. 1A) (1, 2). The nitrogenase catalyzed reaction is generally thought to have the limiting stoichiometry shown in Eq. 1 (3),This equation conveys one of the most puzzling aspects of nitrogenase function, for it incorporates an obligatory formation of one mole of H 2 per mole of N 2 reduced, which requires the waste of two reducing equivalents and four ATP (1, 2). A kinetic framework for nitrogenase function that incorporates the stoichiometry of Eq. 1 is provided by the Lowe-Thorneley model (1, 2, 4), which describes transformations among catalytic intermediates (denoted E n ), where n is the number of electrons and protons (n = 0-8) delivered to MoFe protein. N 2 reduction requires activation of the MoFe protein to the E 4 state in which FeMo-cofactor has accumulated four electrons and four protons stored as two hydrides that bridge Fe atoms [Fe-H-Fe] and two protons presumably bound to sulfides of FeMo-cofactor (Fig. 1B) (5-8). The binding of N 2 to the E 4 state is coupled to the stoichiometric evolution of one H 2 per N 2 reduced.We recently presented a draft mechanism for N 2 reduction by nitrogenase that incorporates a mechanistic explanation for obligatory, reversible H 2 loss upon N 2 binding (5). We considered two models for this process, both built on our characterization of the key E 4 state, and tested them against the numerous constraints imposed by turnover under N 2 plus D 2 or T 2 (5, 9-16). In particular, these constraints include the key findings that during catalytic reduction of N 2 (see Scheme S1), a molecule of D 2 or T 2 will reduce two protons to form two HD or HT without D + /T + exchange with solvent, even though neither D 2 nor T 2 by themselves reacts with nitrogenase during turnover under Ar (5). One model, involving protonation of one of the hydrides to form H 2 , and its replacement by N 2 , was shown to violate ...

N2 reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe-H-Fe] bridging hydrides (denoted E4(4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the N≡N triple bond by the reductive elimination (re) of H2 from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that when E4(4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H2 to give a reactive doubly-reduced intermediate, denoted E4(2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H2 preceded N2 binding. Experiments reported here establish that photoinduced re occurs in two steps. Photolysis of E4(4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E4(2H)* + H2]. The experiments, supported by DFT calculation, indicate that the trapped intermediate is an H2 complex on the ground adiabatic potential energy suface that connects E4(4H) with [E4(2H)* + H2]. We suggest this complex, denoted E4(H2; 2H), is a thermally populated intermediate in the catalytically central re of H2 by E4(4H), and that N2 reacts with this complex to complete the activated conversion of [E4(4H) + N2] into [E4(2N2H) + H2].

Early studies in which nitrogenase was freeze-trapped during enzymatic turnover revealed the presence of high-spin (S = 3/2) electron paramagnetic resonance (EPR) signals from the active-site FeMo-cofactor (FeMo-co) in electron-reduced intermediates of the MoFe protein. Historically denoted as 1b and 1c, each of the signals is describable as a fictitious spin system, S′ = 1/2, with anisotropic g′ tensor, 1b with g′ = [4.21, 3.76, ?] and 1c with g′ = [4.69, ∼3.20, ?]. A clear discrepancy between the magnetic properties of 1b and 1c and the kinetic analysis of their appearance during pre-steady-state turnover left their identities in doubt, however. We subsequently associated 1b with the state having accumulated 2[e–/H+], denoted as E2(2H), and suggested that the reducing equivalents are stored on the catalytic FeMo-co cluster as an iron hydride, likely an [Fe–H–Fe] hydride bridge. Intra-EPR cavity photolysis (450 nm; temperature-independent from 4 to 12 K) of the E2(2H)/1b state now corroborates the identification of this state as storing two reducing equivalents as a hydride. Photolysis converts E2(2H)/1b to a state with the same EPR spectrum, and thus the same cofactor structure as pre-steady-state turnover 1c, but with a different active-site environment. Upon annealing of the photogenerated state at temperature T = 145 K, it relaxes back to E2(2H)/1b. This implies that the 1c signal comes from an E2(2H) hydride isomer of E2(2H)/1b that stores its two reducing equivalents either as a hydride bridge between a different pair of iron atoms or an Fe–H terminal hydride.