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The nitrogenase active-site cofactor must accumulate 4e - /4H + (E 4 (4H) state) before N 2 can bind and be reduced. Earlier studies demonstrated that this E 4 (4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N 2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H 2 . The state having acquired 2e - /2H + (E 2 (2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e - /H + (E 1 (H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E 1 (H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E 1 (H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity , 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E 1 (H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E 1 (H)* relaxing to E 1 (H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E 1 (H)/E 1 (H)* photointerconversion. These observations indicate that the addition of the first e - /H + to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

Mo nitrogenase (N2ase) utilizes a two-component protein system, the catalytic MoFe and its electron-transfer partner FeP, to reduce atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ). The FeMo cofactor contained in the MoFe protein serves as the catalytic center for this reaction and has long inspired model chemistry oriented toward activating N 2 . This field of chemistry has relied heavily on the detailed characterization of how Mo N2ase accomplishes this feat. Understanding the reaction mechanism of Mo N2ase itself has presented one of the most challenging problems in bioinorganic chemistry because of the ephemeral nature of its catalytic intermediates, which are difficult, if not impossible, to singly isolate. This is further exacerbated by the near necessity of FeP to reduce native MoFe, rendering most traditional means of selective reduction inept. We have now investigated the first fundamental intermediate of the MoFe catalytic cycle, E 1 , as prepared both by low-flux turnover and radiolytic cryoreduction, using a combination of Mo Kα high-energy-resolution fluorescence detection and Fe K-edge partial-fluorescence-yield X-ray absorption spectroscopy techniques. The results demonstrate that the formation of this state is the result of an Fe-centered reduction and that Mo remains redox-innocent. Furthermore, using Fe X-ray absorption and 57 Fe Mössbauer spectroscopies, we correlate a previously reported unique species formed under cryoreducing conditions to the natively formed E 1 state through annealing, demonstrating the viability of cryoreduction in studying the catalytic intermediates of MoFe.

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 E 2 (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 E 2 (2H)/1b state now corroborates the identification of this state as storing two reducing equivalents as a hydride. Photolysis converts E 2 (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 E 2 (2H)/1b. This implies that the 1c signal comes from an E 2 (2H) hydride isomer of E 2 (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.

N 2 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 E 4 (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 H 2 from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that, when E 4 (4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H 2 to give a reactive doubly reduced intermediate, denoted E 4 (2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H 2 preceded N 2 binding. Experiments reported here establish that photoinduced re primarily occurs in two steps. Photolysis of E 4 (4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E 4 (2H)* + H 2 ]. The experiments, supported by DFT calculations, indicate that the trapped intermediate is an H 2 complex on the ground adiabatic potential energy suface that connects E 4 (4H) with [E 4 (2H)* + H 2 ]. We suggest that this complex, denoted E 4 (H 2 ; 2H), is a thermally populated intermediate in the catalytically central re of H 2 by E 4 (4H) and that N 2 reacts with this complex to complete the activated conversion of [E 4 (4H) + N 2 ] into [E 4 (2N2H) + H 2 ].

The reduction of N2 to NH3 by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-bound hydrides with obligatory loss of H2 to activate the enzyme for binding/reduction of N2. Earlier work showed that wild-type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO2 by two or eight electrons/protons to carbon monoxide (CO) and methane (CH4) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO2 by two electrons/protons to formate (HCOO(-)) at rates >10 times higher than rates of CO2 reduction to CO and CH4. Quantum mechanical calculations on the doubly reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E2(2H) state) favor a direct reaction of CO2 with the hydride ("direct hydride transfer" reaction pathway), with facile hydride transfer to CO2 yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO2 with the hydride ("associative" reaction pathway), which leads to CO and CH4. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-70(Val→Ala), α-195(His→Gln)) are found to significantly alter the distribution of products between formate and CO/CH4.

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1-8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped "hydride" state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.

A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9:homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 35 GHz electron-nuclear double resonance (ENDOR) measurements on 14/15N and 12/13C isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X not equal N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either 14/15N or 13C: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A(14/15NX) < 0.07/0.1 MHz, A(13CX) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = 14/15N/13C/17O, nuclear quadrupole coupling constant e2qQ for X = 14N, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjunction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7" spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g2 are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if aiso(14/15NX) < 0.03-0.07/0.05-0.1 MHz and aiso(13CX) < 0.05-0.1 MHz, compared with calculated values of aiso(14/15NX) = 0.3/0.4 MHz and aiso(13CX) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.