Search

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]

Investigations of reduction of nitrite (NO2–) to ammonia (NH) by nitrogenase indicate a limiting stoichiometry, NO2– + 6e– + 12ATP + 7H+ → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2– turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2– reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2– reductions in light of their common reduction intermediates and of NO2– reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2– reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e–/H+] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M–[NO+]; transfer of the E2 hydride to the [NO+] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M–[NO] thermodynamic “sink”. The N2 and NO2– reduction pathways converge upon reduction of NH2NH2 and NH2OH bound states to form state H with [−NH2] bound to M. Final reduction converts H to I, with NH bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH occurs upon delivery of five [e–/H+] to N2, but a total of seven [e–/H+] to FeMo-co when obligate H2 evolution is considered, and not earlier in the reduction process. [ABSTRACT FROM AUTHOR]

Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe-H-Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would "waste" two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 bound to FeMo-cofactor 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 D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H+ with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism. [ABSTRACT FROM AUTHOR]

Nitrogenase catalyzes the reduction of N2 and protons to yield two NH3 and one H2. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH3), or hydrazine as substrate recently was shown to trap a common S=½ intermediate, designated I. It was further concluded that the two N-atoms of N2 are hydrogenated alternately ("Alternating" (A) pathway). In the present work, Q-band CW EPR and 95Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state "non-Kramers" doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of 14,15N isotopologs plus 1,2H isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the "prompt" alternating reaction pathway represents a draft mechanism for N2 reduction by nitrogenase. [ABSTRACT FROM AUTHOR]

Enzymatic N2 reduction proceeds along a reaction pathway composed of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e-/H+ delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the 'Distal' (D) pathway, a single N of N2 is hydrogenated in three steps until the first NH3 is liberated, and then the remaining nitrido-N is hydrogenated three more times to yield the second NH3. In the 'Alternating' (A) pathway, the two N's instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH3 liberated only during the fifth step. A recent combination of X/Q-band EPR and 15N, 1,2H ENDOR measurements suggested that states trapped during turnover of the α-70Ala/α-195Gln MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [NxHy] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and 14/15N, 1,2H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N2H2, CH3N2H, and N2H4 in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N2H2, CH3N2H, and N2H4 via a common A reaction pathway, and that the same is true for N2 itself, with Fe ion(s) providing the site of reaction. [ABSTRACT FROM AUTHOR]

Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady- state turnover with N2, the S = 3⁄2 EPR signal (g [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = ½, signal (g [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at ≥ 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable ¹H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor. [ABSTRACT FROM AUTHOR]

A high-resolution (1.16 Å) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, 0, 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 ≠ 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↑:3↓) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4↑:2↓ 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. [ABSTRACT FROM AUTHOR]

A major obstacle to understanding the reduction of N2 to NH3 by nitrogenase has been the impossibility of synchronizing electron delivery to the MoFe protein for generation of specific enzymatic intermediates. When an intermediate is trapped without synchronous electron delivery, the number of electrons, n, it has accumulated is unknown. Consequently, the intermediate is untethered from kinetic schemes for reduction, which are indexed by n. We show that a trapped intermediate itself provides a "synchronously prepared" initial state, and its relaxation to the resting state at 253 K, conditions that prevent electron delivery to MoFe protein, can be analyzed to reveal n and the nature of the relaxation reactions. The approach is applied to the "H+/H- intermediate" (A) that appears during turnover both in the presence and absence of N2 substrate. A exhibits an S = 1/2 EPR signal from the active-site iron-molybdenum cofactor (FeMo-co) to which are bound at least two hydrides/protons. A undergoes two-step relaxation to the resting state (C): A → B → C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE ≈ 3-4 (85% D2O). In the context of the Lowe-Thorneley kinetic scheme for N2 reduction, these results provide powerful evidence that H2 is formed in both relaxation steps, that A is the catalytically central state that is activated for N2 binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons. [ABSTRACT FROM AUTHOR]

Methyldiazene (HNNCH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or ²H were used as substrates for the nitrogenase α-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = 1/2 state that has been characterized by EPR and ¹H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before NN bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to NN bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before NN cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in α-195Gln-substituted MoFe protein. [ABSTRACT FROM AUTHOR]

Focuses on the kinetic mechanism of nitrite reduction by electron nuclear double resonance of protons type 2 and 1. Role of type 1 center in the regulation of activation energy; Characteristics of electron transfer from type 1 to type 2 center; Implication of protons in improving water formation.

The article presents a study which examines whether molybdenum (Mo) is involved during the catalytic turnover particularly in hydride binding. The study used electron nuclear double resonance (ENDOR) measurements of the 95Mo that is improved with molybdenum-iron (MoFe) protein to determine KMo for the intermediate state of E4. The result indicates that Mo is not involved in hydride binding of E4 intermediate but the iron (Fe) ions were involved.

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 ].

Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe-H-Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would "waste" two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 bound to FeMo-cofactor 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 D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H(+) with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.

Na(+)-NQR is the entry point for electrons into the respiratory chain of Vibrio cholerae. It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to translocate sodium across the cell membrane. The enzyme is a membrane complex of six subunits that accommodates a 2Fe-2S center and several flavins. Both the oxidized and reduced forms of Na(+)-NQR exhibit a radical EPR signal. Here, we present EPR and ENDOR data that demonstrate that, in both forms of the enzyme, the radical is a flavin semiquinone. In the oxidized enzyme, the radical is a neutral flavin, but in the reduced enzyme the radical is an anionic flavin, where N(5) is deprotonated. By combining results of ENDOR and multifrequency continuous wave EPR, we have made an essentially complete determination of the g-matrix and all major nitrogen and proton hyperfine matrices. From careful analysis of the W-band data, the full g-matrix of a flavin radical has been determined. For the neutral radical, the g-matrix has significant rhombic character, but this is significantly decreased in the anionic radical. The out-of-plane component of the g-matrix and the nitrogen hyperfine matrices are found to be noncoincident as a result of puckering of the pyrazine ring. Two possible assignments of the radical signals are considered. The neutral and anionic forms of the radical may each arise from a different flavin cofactor, one of which is converted from semiquinone to flavohydroquinone, while the other goes from flavoquinone to semiquinone, at almost exactly the same redox potential, during reduction of the enzyme. Alternatively, both forms of the radical signal may arise from a single, extremely stable, flavin semiquinone, which becomes deprotonated upon reduction of the enzyme.