RESUMO
SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.
Assuntos
Bicamadas Lipídicas/química , Pulmão/química , Proteína B Associada a Surfactante Pulmonar/química , Animais , Bovinos , Bicamadas Lipídicas/metabolismo , Pulmão/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteína B Associada a Surfactante Pulmonar/metabolismo , Relação Estrutura-AtividadeRESUMO
Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O2(-) to H(2)O(2) and O(2). The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band (14)N,(1/2)H continuous wave (CW) and pulsed electron-nuclear double resonance (ENDOR) and X-band (14)N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band (14)N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N(delta) of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band (14)N ESEEM characterized the hyperfine coupling of N(epsilon) of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N(epsilon)-H and O(Glu-17) present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band (1)H CW ENDOR and pulsed (2)H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N(delta) or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.
Assuntos
Níquel/química , Superóxido Dismutase/química , Espectroscopia de Ressonância de Spin Eletrônica , Conformação Molecular , Níquel/metabolismo , Streptomyces/enzimologia , Superóxido Dismutase/isolamento & purificação , Superóxido Dismutase/metabolismoRESUMO
SP-B(CTERM), a cationic, helical peptide based on the essential lung surfactant protein B (SP-B), retains a significant fraction of the function of the full-length protein. Solid-state (2)H- and (31)P-NMR were used to examine the effects of SP-B(CTERM) on mechanically oriented lipid bilayer samples. SP-B(CTERM) modified the multilayer structure of bilayers composed of POPC, POPG, POPC/POPG, or bovine lipid extract surfactant (BLES), even at relatively low peptide concentrations. The (31)P spectra of BLES, which contains approximately 1% SP-B, and POPC/POPG with 1% SP-B(CTERM), look very similar, supporting a similarity in lipid interactions of SP-B(CTERM) and its parent protein, full-length SP-B. In the model systems, although the peptide interacted with both the oriented and unoriented fractions of the lipids, it interacted differently with the two fractions, as demonstrated by differences in lipid headgroup structure induced by the peptide. On the other hand, although SP-B(CTERM) induced similar disruptions in overall bilayer orientation in BLES, there was no evidence of lipid headgroup conformational changes in either the oriented or the unoriented fractions of the BLES samples. Notably, in the model lipid systems the peptide did not induce the formation of small, rapidly tumbling lipid structures, such as micelles, or of hexagonal phases, the observation of which would have provided support for functional mechanisms involving peptide-induced lipid flip-flop or stabilization of curved lipid structures, respectively.
Assuntos
Bicamadas Lipídicas/química , Proteína B Associada a Surfactante Pulmonar/química , Animais , Bovinos , Deutério , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Isótopos de Fósforo , Proteínas Associadas a Surfactantes Pulmonares/químicaRESUMO
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 = 1/2, 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 > or = 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.
Assuntos
Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogênio/química , Nitrogênio/metabolismo , Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Estabilidade Enzimática , Congelamento , Concentração de Íons de Hidrogênio , OxirreduçãoRESUMO
The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure-inducing conditions: 40% hexafluoro-2-propanol aqueous solution at 5 degrees C. Under these conditions it was possible to detect medium-range NOEs for the first time for this peptide. This allowed a much better-defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4-8 of Peptide T take on a well-defined structure with a heavy atom RMSD of 0.78 A. The structure is stabilized by hydrogen bonding to side-chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic gamma-turn.
Assuntos
Peptídeo T/química , Propanóis/química , Estrutura Terciária de Proteína , Solventes/química , Sequência de Aminoácidos , Temperatura Baixa , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeo T/genéticaRESUMO
We extend earlier studies of the effects of relaxation on the intensities of pulsed ENDOR signals by introducing a Davies/Hahn (D/H) pulsed ENDOR multi-sequence that corresponds to a series of Davies sequences with the preparation pulse 'turned off'. In this pulse train, the Hahn [pi/2, pi] detection pulse pair of sequence n-1 both generates the echo detected for that sequence and acts as the preparation portion of sequence n, in effect replacing the pi preparation pulse of the Davies sequence. We show both theoretically, through a master-equation approach, and with both (1)H(I=1/2) and (14)N(I=1) ENDOR experiments on the non-heme Fe enzymes, superoxide reductase (SOR) (S=1/2) and AntDO (S=3/2), that under conditions of high electron-spin polarization (high microwave frequency/low temperature) the D/H multi-sequence allows simplification of ENDOR spectra by suppression of nuclear transitions associated with the m(S)=+1/2 (alpha) manifold. As such suppression depends on the sign of A, it allows determination of this sign. The suppression as a function of the time between individual sequences is found to exhibit behaviors that can be classified into three regimes of the ratio of cross-relaxation to spin-lattice relaxation rates: strong cross-relaxation (X-case); comparable rates (XL); negligible cross relaxation (L). Interestingly, the ENDOR behavior of the S=1/2 SOR center indicates it is an L case, while the S=3/2 AntDO is an L case. Overall, the D/H protocol appears to be a robust and general tool for using relaxation effects to manipulate ENDOR spectra.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ferroproteínas não Heme/química , Oxirredutases/química , Modelos Moleculares , Modelos TeóricosRESUMO
Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N2 reduction, is inhibited by H2. This contrasts with the absence of H2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with alpha-195His substituted by Gln and alpha-70Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g1 = 2.09, g2 = 2.01, and g3 approximately 1.98. 15N- and 1H-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N2 by nitrogenase. Implications of these findings for the mechanism of N2 reduction by nitrogenase are discussed.
Assuntos
Imidas/metabolismo , Nitrogenase/metabolismo , Amônia/análise , Amônia/metabolismo , Cinética , Modelos Moleculares , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogenase/química , Oxirredução , Conformação Proteica , Especificidade por SubstratoRESUMO
A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, 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 not equal 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 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow 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.
Assuntos
Carbono/química , Ferro/química , Molibdênio/química , Nitrogênio/química , Nitrogenase/metabolismo , Enxofre/química , Ferro/metabolismo , Modelos Moleculares , Conformação Molecular , Molibdênio/metabolismoRESUMO
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 alpha-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 alpha-195Gln-substituted MoFe protein.
Assuntos
Imidas/química , Imidas/metabolismo , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Imidas/síntese química , Modelos Moleculares , Nitrogênio/química , Oxirredução , Estrutura Terciária de Proteína , PrótonsRESUMO
Superoxide reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo-Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial Fe-CN and Fe-S bonding is probed by 13C ENDOR of the cyanide ligand and 1Hbeta ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the Fe-C-N linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the spin density on the sulfur in both active sites to a degree that exceeds the difference induced by the alternative sets of "in-plane" nitrogen ligands.
Assuntos
Cânfora 5-Mono-Oxigenase/química , Elétrons , Compostos Férricos/química , Modelos Químicos , Oxirredutases/química , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.
Assuntos
Cianetos/química , Cianetos/metabolismo , Ferroproteínas não Heme/metabolismo , Oxirredutases/metabolismo , Sítios de Ligação , Dicroísmo Circular , Desulfovibrio vulgaris/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Modelos Moleculares , Ferroproteínas não Heme/química , Oxirredutases/química , Pyrococcus furiosus/enzimologia , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Superóxidos/química , Superóxidos/metabolismoRESUMO
EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.
Assuntos
Adamantano/análogos & derivados , Cânfora 5-Mono-Oxigenase/química , Compostos Férricos/química , Compostos Ferrosos/química , Adamantano/química , Adamantano/metabolismo , Sítios de Ligação , Cânfora/análogos & derivados , Cânfora/metabolismo , Cânfora 5-Mono-Oxigenase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Hidroxilação , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.
Assuntos
Molibdoferredoxina/química , Nitrogenase/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Modelos MolecularesRESUMO
We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.
Assuntos
Cânfora 5-Mono-Oxigenase/química , Cânfora 5-Mono-Oxigenase/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Alanina/genética , Alanina/metabolismo , Cânfora 5-Mono-Oxigenase/genética , Isótopos de Carbono , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Flúor/análise , Flúor/química , Heme/química , Heme/metabolismo , Ferro/química , Ferro/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Mutação/genética , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Solventes/química , Especificidade por Substrato , Treonina/genética , Treonina/metabolismoRESUMO
Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.
Assuntos
Acetileno/química , Monóxido de Carbono/química , Elétrons , Molibdoferredoxina/química , Nitrogenase/química , Azotobacter vinelandii/enzimologia , Sítios de Ligação , Etilenos/química , Ferro/química , Cinética , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.
Assuntos
Hidrazinas/química , Nitrogenase/química , Nitrogenase/metabolismo , Azotobacter vinelandii/enzimologia , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Inibidores Enzimáticos/química , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogênio/química , Nitrogênio/metabolismo , Nitrogenase/antagonistas & inibidores , Oxirredução , Prótons , Especificidade por SubstratoRESUMO
A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.
Assuntos
Amidas/química , Aminas/química , Molibdoferredoxina/metabolismo , Nitrogênio/química , Nitrogenase/metabolismo , Sítios de Ligação , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Molibdoferredoxina/química , OxirreduçãoRESUMO
The active site of the oxygenase component of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different alpha-subunits in the (alphabeta)(3) structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates in addition to numerous other adventitious oxidation reactions. High-resolution Mims (2)H-ENDOR spectra have been recorded for the NO-ferrous center of NDO bound with d(8)-naphthalene and d(2)-naphthalene; spectra were collected for the enzyme with the Rieske diiron center both in its oxidized and in its reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of substrate was detected for each substrate. Examination of the sample prepared with 1,4-dideutero-naphthalene shows that the signal arises from D1. The ENDOR data place D1 at a distance of ca. 4.4 A from the mononuclear Fe and with the Fe-D vector being roughly along the Fe-N(O) direction. Because reduction of the Rieske cluster is required for O(2) binding and subsequent catalysis, the effect of its oxidation state on substrate binding was examined. The spectra from the NDO-naphthalene complex reveal two different binding conformations, which change in relative population when the oxidation state of the Rieske cluster is changed. This shift, and the conformational coupling it implies, may hold the key to both oxygen gating and oxygen reactivity for Rieske aromatic dioxygenases.
Assuntos
Proteínas Ferro-Enxofre/química , Complexos Multienzimáticos/química , Oxigenases/química , Dioxigenases , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hidroxilação , Proteínas Ferro-Enxofre/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredução , Oxigenases/metabolismo , Pseudomonas/enzimologiaRESUMO
The active site of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different alpha-subunits in the (alphabeta)(3) structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates, in addition to numerous other adventitious oxidation reactions. High-resolution Mims (2)H-ENDOR (electron nuclear double resonance) spectra have been recorded for the NO-ferrous center of NDO bound with the substrates d(8)-naphthalene, d(2)-naphthalene, d(8)-toluene, d(3)-toluene, and d(6)-benzene; samples were prepared in a D(2)O buffer to test for solvent-derived ligands; spectra were collected for enzymes with the Rieske diiron center in both its oxidized and reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of the substrate in a major binding geometry (denoted as A) was detected for all perdeuterated substrates. Examination of the sample prepared with 1,4-di-deutero-naphthalene shows that the signal arises from D1. Analysis of two-dimensional (2-D) orientation-selective ENDOR patterns collected for this sample defined the location of the D1 deuteron, with respect to the g-frame of the iron center and the orientation of the C-D1 bond. Consideration of the orientations of naphthalene that are permitted within the constraints of these results, as supported by a novel approach to simulations of orientation-selective, 2-D ENDOR patterns for the perdeuterated naphthalene sample, which summed contributions from D1/D2/D8, disclose the geometry of the naphthalene and the Fe-NO fragment. The two deuterons of the reactive carbons, D1 and D2, are closest to the Fe atom (r(Fe)(-)(D1) approximately 4.3 A, r(Fe)(-)(D2) approximately 5.0 A), whereas D8 is farther away (r(Fe)(-)(D8) approximately 5.3 A). Perhaps more instructive, D1-N and D2-N distances to the O(2) surrogate, NO, are approximately 2.4 and approximately 3.3 A, respectively, whereas the D8-N distance is approximately 3.7 A. The data show that benzene and the aromatic ring of toluene also sit within the substrate-binding pocket adjacent to the mononuclear Fe atom. These rings occupy a position similar to that of the "proximal" ring of naphthalene, with the closest ring deuteron being located at a distance of approximately 4.3-4.4 A from the Fe atom and with the Fe-D vector being slightly off the Fe-N(O) direction. In particular, comparison of the data for d(8)-toluene and methyl-d(3)-toluene shows that the methyl group of toluene points away from the Fe atom, despite observations that the oxidation of toluene occurs at the methyl group during catalysis. The Rieske cluster is reduced during both steady-state and single-turnover catalysis; therefore, the effect of its oxidation state on the geometry of substrate binding was examined. The spectra from the NDO-naphthalene complex also revealed a second binding conformation (denoted as B), in which the substrate is located approximately 0.5 A farther from the Fe atom. The relative populations of A- and B-sites are allosterically changed when the Rieske cluster is reduced. ENDOR of exchangeable protons shows that the water/hydroxide of Fe-NDO is retained upon binding NO.
Assuntos
Proteínas Ferro-Enxofre/química , Complexos Multienzimáticos/química , Oxigenases/química , Deutério , Dioxigenases , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Ferrosos/química , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Oxigenases/metabolismoRESUMO
Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.