RESUMEN
[FeFe]-hydrogenses and molybdenum (Mo)-nitrogenase are evolutionarily unrelated enzymes with unique complex iron-sulfur cofactors at their active sites. The H cluster of [FeFe]-hydrogenases and the FeMo cofactor of Mo-nitrogenase require specific maturation machinery for their proper synthesis and insertion into the structural enzymes. Recent insights reveal striking similarities in the biosynthetic pathways of these complex cofactors. For both systems, simple iron-sulfur cluster precursors are modified on assembly scaffolds by the activity of radical S-adenosylmethionine (SAM) enzymes. Radical SAM enzymes are responsible for the synthesis and insertion of the unique nonprotein ligands presumed to be key structural determinants for their respective catalytic activities. Maturation culminates in the transfer of the intact cluster assemblies to a cofactor-less structural protein recipient. Required roles for nucleotide binding and hydrolysis have been implicated in both systems, but the specific role for these requirements remain unclear. In this review, we highlight the progress on [FeFe]-hydrogenase H cluster and nitrogenase FeMo-cofactor assembly in the context of these emerging paradigms.
Asunto(s)
Bacterias/metabolismo , Coenzimas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Azufre/metabolismo , Bacterias/enzimología , Región Branquial/enzimología , Región Branquial/metabolismo , Coenzimas/química , Hidrogenasas/química , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/química , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Nitrogen-fixing bacteria catalyze the reduction of dinitrogen (N(2)) to two ammonia molecules (NH(3)), the major contribution of fixed nitrogen to the biogeochemical nitrogen cycle. The most widely studied nitrogenase is the molybdenum (Mo)-dependent enzyme. The reduction of N(2) by this enzyme involves the transient interaction of two component proteins, designated the iron (Fe) protein and the MoFe protein, and minimally requires 16 magnesium ATP (MgATP), eight protons, and eight electrons. The current state of knowledge on how these proteins and small molecules together effect the reduction of N(2) to ammonia is reviewed. Included is a summary of the roles of the Fe protein and MgATP hydrolysis, information on the roles of the two metal clusters contained in the MoFe protein in catalysis, insights gained from recent success in trapping substrates and inhibitors at the active-site metal cluster FeMo cofactor, and finally, considerations of the mechanism of N(2) reduction catalyzed by nitrogenase.
Asunto(s)
Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Bacterias/enzimología , Bacterias/metabolismo , Molibdoferredoxina/química , Fijación del Nitrógeno , Nitrogenasa/química , Nitrogenasa/genéticaRESUMEN
Nitrogenases utilize Fe-S clusters to reduce N2 to NH3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+ results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.
Asunto(s)
Azotobacter vinelandii/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Precursores de Proteínas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de MossbauerRESUMEN
Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe7S8C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe3S3C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M'Fe3S3X]n-, where M' = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M' results in minor changes, the chelating, µ3-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.
Asunto(s)
Hierro/metabolismo , Molibdeno/metabolismo , Molibdoferredoxina/química , Carbamatos/química , Carbamatos/metabolismo , Carbono/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hierro/química , Ligandos , Modelos Moleculares , Estructura Molecular , Molibdeno/química , Molibdoferredoxina/metabolismo , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Nitrogenasa/metabolismo , Oxidación-Reducción , Sulfuros/química , Sulfuros/metabolismo , Azufre/metabolismoRESUMEN
Nitrogenase MoFe protein can be coupled with CdS nanocrystals (NCs) to enable photocatalytic N2 reduction. The nature of interactions that support complex formation is of paramount importance in intermolecular electron transfer that supports catalysis. In this work we have employed microscale thermophoresis to examine binding interactions between 3-mercaptopropionate capped CdS quantum dots (QDs) and MoFe protein over a range of QD diameters (3.4-4.3 nm). The results indicate that the interactions are largely electrostatic, with the strength of interactions similar to that observed for the physiological electron donor. In addition, the strength of interactions is sensitive to the QD diameter, and the binding interactions are significantly stronger for QDs with smaller diameters. The ability to quantitatively assess NC protein interactions in biohybrid systems supports strategies for understanding properties and reaction parameters that are important for obtaining optimal rates of catalysis in biohybrid systems.
Asunto(s)
Molibdoferredoxina , Puntos Cuánticos , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Electricidad Estática , Nitrogenasa/química , Nitrogenasa/metabolismo , Transporte de ElectrónRESUMEN
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.
Asunto(s)
Azotobacter vinelandii , Nitrogenasa , Azotobacter vinelandii/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Molibdoferredoxina/metabolismo , Nitrogenasa/genética , Nitrogenasa/metabolismoRESUMEN
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.
Asunto(s)
Molibdoferredoxina , Nitrogenasa , Nitrogenasa/química , Nitrogenasa/metabolismo , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Sustitución de Aminoácidos , Oxidación-Reducción , Conformación Molecular , Aminoácidos , ProtonesRESUMEN
Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction. The molybdenum-iron nitrogenase is the most well-studied, but redox potentials associated with proton-coupled transformations between the redox levels of the catalytic MoFe protein have proved difficult to pin down, in part due to a complex electron-transfer pathway from the partner Fe protein, linked to ATP-hydrolysis. Here, we apply electrochemical control to the MoFe protein of Azotobacter vinelandii nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators. We combine insight from the electrochemical current response with data from gas chromatography and in situ infrared spectroscopy, in order to define potentials for the binding of a series of inhibitors (carbon monoxide, methyl isocyanide) to the metallo-catalytic site of the MoFe protein, and the onset of catalytic transformation of alternative substrates (protons and acetylene) by the enzyme. Thus, we associate potentials with the redox levels for inhibition and catalysis by nitrogenase, with relevance to the elusive mechanism of biological nitrogen fixation.
Asunto(s)
Molibdoferredoxina , Nitrogenasa , Nitrogenasa/química , Nitrogenasa/metabolismo , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Oxidación-Reducción , Proteínas/metabolismo , Fijación del NitrógenoRESUMEN
The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.
Asunto(s)
Azotobacter vinelandii , Puntos Cuánticos , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Temperatura , Oxidación-Reducción , Nitrogenasa/química , Nitrogenasa/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Azotobacter vinelandii/metabolismoRESUMEN
A conductive polymer-based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non-photosynthetic bacterium Azotobacter Chroococcum (A.â Chroococcum). The light-harvesting cationic poly(fluorene-alt-phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4 + -N and L-amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum-iron (MoFe) protein and relevant nitrogen-fixing proteins are up-regulated. These photoactive conductive polymer-bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non-photosynthetic nitrogen-fixing bacteria.
Asunto(s)
Fijación del Nitrógeno , Nitrogenasa , Nitrogenasa/metabolismo , Molibdoferredoxina/metabolismo , Hierro/metabolismo , Oxidación-ReducciónRESUMEN
Mo-nitrogenase catalyzes the challenging N2-to-NH3 reduction. This complex reaction proceeds through a series of intermediate states (En) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between En states is central to defining the mechanism of nitrogenase. Here, rate constants of key steps have been determined through a steady-state kinetic model with fits to experimental data. The model reveals that the rate for H2 formation from the early electron populated state E2(2H) is much slower than that from the more reduced E4(4H) state. Further, it is found that the competing reactions of H2 formation and N2 binding at the E4(4H) state occur with equal rate constants. The H2-dependent reverse reaction of the N2 binding step is found to have a rate constant of 5.5 ± 0.2 (atm H2)-1 s-1 (7.2 ± 0.3 (mM H2)-1 s-1). Importantly, the reduction of N2 bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s-1, revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical transformation of N2 to 2 NH3. Finally, the populations of En states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.
Asunto(s)
Molibdoferredoxina , Nitrogenasa , Catálisis , Cinética , Molibdoferredoxina/metabolismo , Nitrogenasa/química , Oxidación-ReducciónRESUMEN
The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer. We report the distribution of ethylene, dihydrogen, and methane isotopologue products measured from nitrogenase-catalyzed reductions of acetylene, protons, and cyanide, respectively, performed in varying levels of deuterium enrichment of the solvent. As has been noted previously, the total rate of product formation by nitrogenase is largely insensitive to the presence of D2O in the solvent. Nevertheless, the incorporation of H/D into products can be measured for these substrates that reflect solvent isotope effects on hydrogen atom transfers that are faster than the overall rate-determining step for nitrogenase. From these data, a minimal isotope effect is observed for acetylene reduction (1.4 ± 0.05), while the isotope effects for hydrogen and methane evolution are significantly higher at 4.2 ± 0.1 and 4.4 ± 0.1, respectively. These results indicate that there are pronounced differences in the sensitivity to isotopic substitution of the hydrogen atom transfer steps associated with the reduction of these substrates by nitrogenase.
Asunto(s)
Azotobacter vinelandii , Nitrogenasa , Nitrogenasa/metabolismo , Molibdoferredoxina/metabolismo , Deuterio/metabolismo , Protones , Solventes , Oxidación-Reducción , Acetileno , Hidrógeno/metabolismo , Metano/metabolismoRESUMEN
There is an evergrowing demand for environment-friendly processes to synthesize ammonia (NH3) from atmospheric nitrogen (N2). Although diazotrophic N2 fixation represents an undeniably "green" process of NH3 synthesis, the slow reaction rate makes it less suitable for industrially meaningful large-scale production. Here, we report the photoinduced N2 fixation using a hybrid system composed of colloidal quantum dots (QDs) and aerobic N2-fixing bacteria, Azotobacter vinelandii. Compared to the case where A. vinelandii cells are simply mixed with QDs, NH3 production increases significantly when A. vinelandii cells are cultured in the presence of core/shell InP/ZnSe QDs. During the cell culture of A. vinelandii, the cellular uptake of QDs is facilitated in the exponential growth phase. Experimental results as well as theoretical calculations indicate that the photoexcited electrons in QDs within A. vinelandii cells are directly transferred to MoFe protein, the catalytic component of nitrogenase. We also observe that the excess amount of QDs left on the outer surface of A. vinelandii disrupts the cellular membrane, leading to the decrease in NH3 production due to the deactivation of nitrogenase. The successful uptake of QDs in QD-A. vinelandii hybrid with minimal amount of QDs on the outer surface of the bacteria is key to efficient photosensitized NH3 production. The comprehensive understanding of the QD-bacteria interface paves an avenue to novel and efficient nanobiohybrid systems for chemical production.
Asunto(s)
Azotobacter vinelandii , Puntos Cuánticos , Amoníaco/metabolismo , Azotobacter vinelandii/metabolismo , Bacterias/metabolismo , Molibdoferredoxina/metabolismo , Fijación del Nitrógeno , Nitrogenasa/metabolismoRESUMEN
The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue ß-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing ß-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing ß-188Ser) are important for communication between the two halves.
Asunto(s)
Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Azotobacter vinelandii/enzimología , Sitios de Unión , Medición de Intercambio de Deuterio , Transporte de Electrón , Modelos Moleculares , Unión ProteicaRESUMEN
Inspired by mechanistic proposals for N2 reduction at the nitrogenase FeMo cofactor, we report herein a new, strongly σ-donating heteroscorpionate ligand featuring two weak-field pyrazoles and an alkyl donor. This ligand supports four-coordinate Fe(I)-N2, Fe(II)-Cl, and Fe(III)-imido complexes, which we have characterized using a variety of spectroscopic and computational methods. Structural and quantum mechanical analysis reveal the nature of the Fe-C bonds to be essentially invariant between the complexes, with conversion between the (formally) low-valent Fe-N2 and high-valent Fe-imido complexes mediated by pyrazole hemilability. This presents a useful strategy for substrate reduction at such low-coordinate centers and suggests a mechanism by which FeMoco might accommodate the binding of both π-acidic and π-basic nitrogenous substrates.
Asunto(s)
Compuestos Férricos , Molibdoferredoxina , Compuestos Férricos/metabolismo , Ligandos , Modelos Moleculares , Molibdoferredoxina/metabolismo , Oxidación-ReducciónRESUMEN
CO-bound forms of nitrogenase are N2-reduction inhibited and likely intermediates in Fischer-Tropsch chemistry. Visible-light photolysis at 7 K was used to interrogate all three known CO-related EPR-active forms as exhibited by the α-H195Q variant of Azotobacter vinelandii nitrogenase MoFe protein. The hi(5)-CO EPR signal converted to the hi-CO EPR signal, which reverted at 10 K. FT-IR monitoring revealed an exquisitely light-sensitive "Hi-2" species with bands at 1932 and 1866 cm-1 that yielded "Hi-1" with bands at 1969 and 1692 cm-1, which reverted to "Hi-2". The similarities of photochemical behavior and recombination kinetics showed, for the first time, that hi-CO EPR and "Hi-1" IR signals arise from one chemical species. hi(5)-CO EPR and "Hi-2" IR signals are from a second species, and lo-CO EPR and "Lo-2" IR signals, formed after prolonged illumination, are from a third species. Comparing FT-IR data with CO-inhibited MoFe-protein crystal structures allowed assignment of CO-bonding geometries in these species.
Asunto(s)
Azotobacter vinelandii , Nitrogenasa , Monóxido de Carbono , Espectroscopía de Resonancia por Spin del Electrón , Molibdoferredoxina/metabolismo , Nitrogenasa/química , Recombinación Genética , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The nitrogenase active-site cofactor must accumulate 4e-/4H+ (E4(4H) state) before N2 can bind and be reduced. Earlier studies demonstrated that this E4(4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H2. The state having acquired 2e-/2H+ (E2(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e-/H+ (E1(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E1(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E1(H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity, 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E1(H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E1(H)* relaxing to E1(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E1(H)/E1(H)* photointerconversion. These observations indicate that the addition of the first e-/H+ to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.
Asunto(s)
Electrones , Nitrogenasa , Espectroscopía de Resonancia por Spin del Electrón , Hidrógeno/química , Metales/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenasa/química , Oxidación-ReducciónRESUMEN
Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 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 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca 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 E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 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.
Asunto(s)
Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Azotobacter vinelandii/enzimología , Isótopos de Carbono , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Marcaje Isotópico , Conformación Molecular , Molibdoferredoxina/química , Nitrogenasa/química , Nitrogenasa/aislamiento & purificaciónRESUMEN
The iron-molybdenum cofactor (FeMoco) is responsible for dinitrogen reduction in Mo nitrogenase. Unlike the resting state, E0 , reduced states of FeMoco are much less well characterized. The E2 state has been proposed to contain a hydride but direct spectroscopic evidence is still lacking. The E2 state can, however, relax back the E0 state via a H2 side-reaction, implying a hydride intermediate prior to H2 formation. This E2 âE0 pathway is one of the primary mechanisms for H2 formation under low-electron flux conditions. In this study we present an exploration of the energy surface of the E2 state. Utilizing both cluster-continuum and QM/MM calculations, we explore various classes of E2 models: including terminal hydrides, bridging hydrides with a closed or open sulfide-bridge, as well as models without. Importantly, we find the hemilability of a protonated belt-sulfide to strongly influence the stability of hydrides. Surprisingly, non-hydride models are found to be almost equally favorable as hydride models. While the cluster-continuum calculations suggest multiple possibilities, QM/MM suggests only two models as contenders for the E2 state. These models feature either i) a bridging hydride between Fe2 and Fe6 and an open sulfide-bridge with terminal SH on Fe6 (E2 -hyd) or ii) a double belt-sulfide protonated, reduced cofactor without a hydride (E2 -nonhyd). We suggest both models as contenders for the E2 redox state and further calculate a mechanism for H2 evolution. The changes in electronic structure of FeMoco during the proposed redox-state cycle, E0 âE1 âE2 âE0 , are discussed.
Asunto(s)
Molibdoferredoxina , Nitrogenasa , Electrones , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Oxidación-ReducciónRESUMEN
Carbon monoxide (CO) is a well-known inhibitor of nitrogenase activity. Under turnover conditions, CO binds to FeMoco, the active site of Mo nitrogenase. Time-resolved IR measurements suggest an initial terminal CO at 1904 cm-1 that converts to a bridging CO at 1715 cm-1, and an X-ray structure shows that CO can displace one of the bridging belt sulfides of FeMoco. However, the CO-binding redox state(s) of FeMoco (En) and the role of the protein environment in stabilizing specific CO-bound intermediates remain elusive. In this work, we carry out an in-depth analysis of the CO-FeMoco interaction based on quantum chemical calculations addressing different aspects of the electronic structure. (1) The local electronic structure of the Fe-CO bond is studied through diamagnetically substituted FeMoco. (2) A cluster model of FeMoco within a polarizable continuum illustrates how CO binding may affect the spin-coupling between the metal centers. (3) A QM/MM model incorporates the explicit influence of the amino acid residues surrounding FeMoco in the MoFe protein. The QM/MM model predicts both a terminal and a bridging CO in the E1 redox state. The scaled calculated CO frequencies (1922 and 1716 cm-1, respectively) are in good agreement with the experimentally observed IR bands supporting CO binding to the E1 state. Alternatively, an E2 state QM/MM model, which has the same atomic structure as the CO-bound X-ray structure, features a semi-bridging CO with a scaled calculated frequency (1718 cm-1) similar to the bridging CO in the E1 model.