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Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data—although rich with information—combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe–9S–C–Mo–homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe–C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited “hi-CO” state; (v) E4(4H) Janus intermediate, activated for N2binding/reduction by accumulation of 4[e–/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S= 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca= [−1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe−C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe–C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe–C bonding of the CFe6core indicate that it does not provide a functionally dynamic (hemilabile) “beating heart”─instead it acts as “a heart of steel”, stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

The nitrogenase active-site cofactor must accumulate 4e–/4H+(E4(4H) state) before N2can bind and be reduced. Earlier studies demonstrated that this E4(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 N2binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H2. The state having acquired 2e–/2H+(E2(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e–/H+(E1(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E1(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E1(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 E1(H)*) with g= [2.009, 1.950, 1.860], which results in a photostationary state, with E1(H)* relaxing to E1(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E1(H)/E1(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 formallyone-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

Mo-dependent nitrogenase is a major contributor to global biological N2reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4−) centered within a trigonal prismatic CFe6core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S= 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca= +0.86 MHz. Turnover under high CO partial pressure generates the S= 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitudeof the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca= −1.30 MHz. This indicates that both the E0and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Cafrom three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6core structures of E0and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide−Fe bonds.

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E4(4H)) with two [Fe–H–Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E4(4H) hydrides showed that enzymatic reof E4(4H) hydride yields an H2-bound complex (E4(H2,2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe–H bonds to the metal ions themselves enables N2to bind with concomitant H2release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E4(H2,2H) has not been trapped, cryogenic photolysis of E4(4H) provides a means to address this question. Photolysis of E4(4H) causes hydride-rewith release of H2, generating doubly reduced FeMo-co (denoted E4(2H)*), the extreme limit of the electron-density redistribution upon formation of E4(H2,2H). Here we examine the doubly reduced FeMo-co core of the E4(2H)* limiting-state by 1H, 57Fe, and 95Mo ENDOR to illuminate the partial electron-density redistribution upon E4(H2,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E4(2H)* ENDOR results as extended by DFT computations include (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; and (ii) two distinctive E4(4H) 57Fe signals are suggested as associated with structurally identified “anchors” of one bridging hydride, two others with identified anchors of the second, with NBO-analysis further identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe–H bonds.

Nitrogenase is activated for N2reduction through the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co: Fe7S9MoC; homocitrate) to form the key Janus intermediate, denoted E4(4H), whose lowest-energy structure contains two [Fe–H–Fe] bridging hydrides and two protons bound to the sulfurs that also bridge the Fe pairs. In the critical step of catalysis, a H2complex transiently produced by reductive elimination (re) of the hydrides of E4(4H), denoted E4(H2;2H), undergoes H2displacement by N2, which then undergoes the otherwise energetically unfavorable cleavage of the N≡N triple bond. In pursuing the study of the reactivation process, we have employed a photochemical approach to obtaining its atomic-level details. Continuous 450 nm irradiation of the ground state of the dihydride Janus intermediate, denoted E4(4H)a, in an EPR cavity at cryogenic temperatures causes photoinduced reof H2to generate E4(H2;2H). We here extend this photochemical approach with time-resolved EPR studies of the photolysis process on the ns time scale. These studies reveal an additional intermediate in the catalytic reductive elimination process, an isomer of the E4(4H) FeMo-co metal-ion core that is formed prior to E4(H2;2H) and is thought to be created by breaking an Fe–SH bond, thus further integrating the calculational and structural studies into the experimentally determined mechanism by which nitrogenase is activated to cleave the N≡N triple bond.

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) (“Janus”) state. N2binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oamechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2rewith N2binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2turnover (and H2inhibition of N2reduction), thereby establishing the re/oaequilibrium as a universal mechanism for N2binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (kHP) for H2production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the reof H2coupled to N2binding/reduction, which decreases in the order Mo > V > Fe.

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.

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N2reduction relative to H2evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe–H–Fe hydrides on the active site FeMo-cofactor to yield H2is a key feature in the N2reduction mechanism. The N2reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2reduction (1 atm) of 181 ± 5 nmol NH3min–1mg–1FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2min–1mg–1FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4min–1mg–1FeFe protein. Under turnover conditions, N2reduction is inhibited by H2and the enzyme catalyzes the formation of HD when presented with N2and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2reduction occurring by reductive elimination of H2.

N2reduction 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 H2from this state. We are exploring a photochemical approach to obtaining atomic-level details of the reactivation 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 reof H2to give a reactive doubly reduced intermediate, denoted E4(2H)*, which corresponds to the intermediate that would form if thermal dissociative reloss of H2preceded N2binding. Experiments reported here establish that photoinduced reprimarily 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 calculations, indicate that the trapped intermediate is an H2complex on the ground adiabatic potential energy suface that connects E4(4H) with [E4(2H)* + H2]. We suggest that this complex, denoted E4(H2; 2H), is a thermally populated intermediate in the catalytically central reof H2by E4(4H) and that N2reacts with this complex to complete the activated conversion of [E4(4H) + N2] into [E4(2N2H) + H2].

The binuclear CuA site engineered into Pseudomonas aeruginosa azurin has provided a CuA-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to CuA, notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine Cβ protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence CuA systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the CuA core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear CuA center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.