Search

A study is conducted to demonstrate that wild-type nitrogenase reduces diazene to ammonia. The findings suggest that the reduction of [N.sub.2] and diazene is inhibited by [H.sub.2].

EPR and ENDOR data, which show conclusively that the radical signals present in the oxidized and reduced forms of Na(super +) -NQR are both flavin semiquinones is presented. Irrespective of whether the neutral and anionic radical signals in Na(super +)-NQR arise from one or two flavins, a flavin semiquinone could play a role in the translocation of sodium ions by the enzyme.

Research has been conducted on the nitrite reductases. The kinetic mechanism of the nitrite reduction and the pH dependence on the enzymatic activity and its activation energy have been investigated and the details are reported.

The possible involvement of Mo in substrate interactions during catalytic turnover is addressed. The response of the Mo coupling to subtle conformational changes in [E.sub.0] and to the formation of [E.sub.4] has shown that Mo is involved in tuning the geometric and electronic properties of FeMo-co in these states.

The intermediate generated at a high concentration during reduction of the natural nitrogenase substrate ([N.sub.2]) by wild-type MoFe protein has shown that it contains [N.sub.2] bound to the active-site FeMo cofactor. The single type of [super 15]N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable [super 1]H ENDOR signal, is shown to be consistent with the [N.sub.2] molecule bound end-on to the FeMo cofactor.

The dipolar hyperfine coupling determined between the (Iron bleomycin) BLM Fe and the 2' substituent of the cytidine sugar where H4' abstraction occurs. Fe-2'F distances may be compared to analogous distances from plausibly relevant NMR-derived Co-BLM-oligonucleotide structures.

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]

The article presents a study on nitrogen fixation mechanism through nitrogenase. It mentions the significant role of the element in sustaining life while contained in biomolecules as well as its availability in the atmosphere as dinitrogen (N2) gas. It also notes the occurrence of fixation in lightning and the Haber-Bosch process.

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 mis 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, 1 b and 1c, trapped under high electron flux. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation, the reduction of N2to two NH3molecules, supports more than half the human population. The predominant form of the enzyme nitrogenase, which catalyzes this reaction, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Although nitrogenase has been studied extensively, the catalytic mechanism has remained unknown. At a minimum, a mechanism must identify and characterize each intermediate formed during catalysis and embed these intermediates within a kinetic framework that explains their dynamic interconversion. The Lowe–Thorneley (LT) model describes nitrogenase kinetics and provides rate constants for transformations among intermediates (denoted En, where nis the number of electrons (and protons), that have accumulated within the MoFe protein). Until recently, however, research on purified nitrogenase had not characterized any Enstate beyond E0. [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]

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.

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.

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-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 ].

We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.

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.

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and NO in a two-step process involving the intermediate N(ω)-hydroxy-L-arginine (NHA). It was shown that Cpd I is the oxygenating species for L-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N(ω)-OH and N(ω)-H of NHA significantly inhibits the conversion of NHA into NO and L-citrulline by mammalian NOS. Kinetic studies now show that N(ω)-methylation of NHA has a qualitatively similar effect on H₂O₂-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N(ω)-hydroxy L-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA's, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of L-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of L-citrulline in these experiments and k(obs) for the H₂O₂-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N(ω)-OH and N(ω)-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.

Investigations of reduction of nitrite (NO2(-)) to ammonia (NH3) 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 NH3 bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH3 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.

Enzymatic N(2) 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 N(2) is hydrogenated in three steps until the first NH(3) is liberated, and then the remaining nitrido-N is hydrogenated three more times to yield the second NH(3). 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 NH(3) liberated only during the fifth step. A recent combination of X/Q-band EPR and (15)N, (1,2)H ENDOR measurements suggested that states trapped during turnover of the α-70(Ala)/α-195(Gln) MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [N(x)H(y)] 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 (14/15)N, (1,2)H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N(2)H(2), CH(3)N(2)H, and N(2)H(4) 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 N(2)H(2), CH(3)N(2)H, and N(2)H(4) via a common A reaction pathway, and that the same is true for N(2) itself, with Fe ion(s) providing the site of reaction.

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.