Search

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e−/H+] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e−/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e−/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e−/H+] generates E4(4H) with Fe2–H–Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E2(2H) containing Fe3–H–Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E2(2H) contains Fe2–H–Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme. [ABSTRACT FROM AUTHOR]

We recently demonstrated that N2 reduction by nitrogenase involves the obligatory release of one H2 per N2 reduced. These studies focus 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 monometallic 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. [ABSTRACT FROM AUTHOR]

Freeze-quenching nitrogenase during turnover with N2 traps an S = 1/2 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 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 with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3. [ABSTRACT FROM AUTHOR]

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e - /H + ] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e - /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e - /H + ] to the E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e - /H + ] generates E 4 (4H) with Fe2-H-Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E 2 (2H) containing Fe3-H-Fe7. In the dominant α-V70I E 4 (4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E 2 (2H) contains Fe2-H-Fe6. It is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme.

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 CFe 6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13 C 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 13 CFe 6 ENDOR measurements for (i) the wild-type protein resting state ( E 0 ; α-Val 70 ) to those of (ii) α-Ile 70 , (iii) α-Ala 70 -substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E 4 (4H) Janus intermediate, activated for N 2 binding/reduction by accumulation of 4[e - /H + ]; (vi) E 4 (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 13 C isotropic hyperfine coupling constants, C a = [-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 CFe 6 Fe-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 CFe 6 core 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.

Mo-dependent nitrogenase is a major contributor to global biological N 2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe 7 MoS 9 C-homocitrate) contains a carbide (C 4- ) centered within a trigonal prismatic CFe 6 core 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 13 C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13 C-carbide ENDOR of the S = 3/2 resting state (E 0 ) is remarkable, with an extremely small isotropic hyperfine coupling constant, C a = +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 magnitude of the 13 C carbide isotropic hyperfine-coupling constant essentially unchanged, C a = -1.30 MHz. This indicates that both the E 0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to C a from 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 E 0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe 6 core structures of E 0 and 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 E 4 (4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination ( re ) of the E 4 (4H) hydrides showed that enzymatic re of E 4 (4H) hydride yields an H 2 -bound complex (E 4 (H 2 ,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 N 2 to bind with concomitant H 2 release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E 4 (H 2 ,2H) has not been trapped, cryogenic photolysis of E 4 (4H) provides a means to address this question. Photolysis of E 4 (4H) causes hydride- re with release of H 2 , generating doubly reduced FeMo-co (denoted E 4 (2H)*), the extreme limit of the electron-density redistribution upon formation of E 4 (H 2 ,2H). Here we examine the doubly reduced FeMo-co core of the E 4 (2H)* limiting-state by 1 H, 57 Fe, and 95 Mo ENDOR to illuminate the partial electron-density redistribution upon E 4 (H 2 ,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E 4 (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 E 4 (4H) 57 Fe 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 N 2 reduction through the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co: Fe7S9MoC; homocitrate) to form the key Janus intermediate, denoted E 4 (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 H 2 complex transiently produced by reductive elimination ( re ) of the hydrides of E 4 (4H), denoted E 4 (H 2 ;2H), undergoes H 2 displacement by N 2 , which then undergoes the otherwise energetically unfavorable cleavage of the N≡N triple bond. In pursuing the study of the re activation 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 E 4 (4H) a , in an EPR cavity at cryogenic temperatures causes photoinduced re of H 2 to generate E 4 (H 2 ;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 E 4 (4H) FeMo-co metal-ion core that is formed prior to E 4 (H 2 ;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 N 2 reduction to 2NH 3 : Mo-, V-, and Fe-nitrogenase, named respectively for the metal ( M ) in their active site metallocofactors (metal-ion composition, M Fe 7 ). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N 2 binding 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 E 4 (4H) ("Janus") state. N 2 binding/reduction in this state is coupled to reductive elimination ( re ) of the two hydrides as H 2 , the forward direction of a reductive-elimination/oxidative-addition ( re/oa ) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N 2 /D 2 . Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H 2 formation by hydride protonolysis ( k HP ) but significant differences in the rate constant for H 2 re with N 2 binding/reduction ( k re ). We now report that V-nitrogenase also exhibits HD formation during N 2 /D 2 turnover (and H 2 inhibition of N 2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N 2 binding 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 ( k HP ) for H 2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant ( k re ) for the re of H 2 coupled to N 2 binding/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 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.

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N 2 reduction relative to H 2 evolution. 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 H 2 is a key feature in the N 2 reduction mechanism. The N 2 reduction 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 N 2 reduction. 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 N 2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N 2 reduction (1 atm) of 181 ± 5 nmol NH 3 min -1 mg -1 FeFe protein, for proton reduction (in the absence of N 2 ) of 1085 ± 41 nmol H 2 min -1 mg -1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C 2 H 4 min -1 mg -1 FeFe protein. Under turnover conditions, N 2 reduction is inhibited by H 2 and the enzyme catalyzes the formation of HD when presented with N 2 and D 2 . 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 N 2 reduction occurring by reductive elimination of H 2 .

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H 2 . A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H 2 . The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H 2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H 2 O and D 2 O, the proton inventory, was found to change linearly with the D 2 O/H 2 O ratio, revealing that a single H/D is involved in the rate-limiting step of H 2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H 2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H 2 formation where the S-H + bond breaks and H + attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H 2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

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 ].