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1.
Acc Chem Res ; 40(7): 475-83, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17567087

RESUMO

Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.


Assuntos
Dioxigenases/química , Ferroproteínas não Heme/química , Oxigênio/metabolismo , Oxigenases/química , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dioxigenases/metabolismo , Humanos , Cinética , Ferroproteínas não Heme/metabolismo , Oxirredução , Oxigênio/química , Oxigenases/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo
2.
J Am Chem Soc ; 123(48): 11831-7, 2001 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-11724588

RESUMO

Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.


Assuntos
Cicloeptanos/química , Oxigenases/química , Cicloeptanos/metabolismo , Hidroxilação , Cinética , Methylosinus trichosporium/enzimologia , Oxirredução , Oxigenases/metabolismo , Solubilidade
3.
J Biol Inorg Chem ; 6(7): 717-25, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11681705

RESUMO

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.


Assuntos
Oxigenases/química , Oxigenases/metabolismo , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Cicloexanos/química , Cicloexanos/metabolismo , Cicloexenos , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Peróxido de Hidrogênio/química , Hidroxilação , Methylosinus trichosporium/enzimologia , NAD/metabolismo , Oxirredução
5.
Biochemistry ; 40(32): 9539-51, 2001 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-11583153

RESUMO

Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.


Assuntos
Metaloproteínas/química , Methylosinus trichosporium/enzimologia , Oxigenases/química , Sequência de Aminoácidos , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Oxigenases/genética , Oxigenases/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
Biochemistry ; 40(7): 2220-33, 2001 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-11329291

RESUMO

Component interactions play important roles in the regulation of catalysis by methane monooxygenase (MMO). The binding of component B (MMOB) to the hydroxylase component (MMOH) has been shown in previous studies to cause structural changes in MMOH that result in altered thermodynamic and kinetic properties during the reduction and oxygen binding steps of the catalytic cycle. Here, specific amino acid residues of MMOB that play important roles in the interconversion of several intermediates of the MMO cycle have been identified. Both of the histidine residues in Methylosinus trichosporium OB3b MMOB (H5 and H33) were chemically modified by diethylpyrocarbonate (DEPC). Although the DEPC--MMOB species exhibited only minor changes relative to unmodified MMOB in steady-state MMO turnover, large decreases in the formation rate constants of the reaction cycle intermediates, compound P and compound Q, were observed. The site specific mutants H5A, H33A, and H5A/H33A were made and characterized. H5A and wild type MMOB elicited similar steady-state and transient kinetics, although the mutant caused a slightly lower rate constant for Q formation. Conversely, H33A exhibited a >50-fold decrease in the P formation rate constant, which resulted in slower formation of Q. The kinetics of the double mutant (H5A/H33A) were similar to those of H33A, suggesting that the highly conserved residue, H33, has the most significant effect on the efficient progress of the cycle. Ongoing NMR investigations of residues perturbed by formation of the MMOH-MMOB complex suggested construction of the MMOB N107G/S109A/S110A/T111A quadruple mutant. This mutant was found to elicit a nearly 2-fold increase in specific activity for steady-state MMO turnover of large substrates such as furan and nitrobenzene but caused no similar increase for the physiological substrate, methane. While the quadruple mutant did not have a significant effect on P and Q formation, it caused an almost 3-fold increase in the decay rate constant of Q for furan oxidation and a 2-fold faster product release rate constant for p-nitrophenol resulting from nitrobenzene oxidation. Conversely, this mutant caused the Q decay rate constant to decrease 7-fold for methane oxidation but left the product release step unaffected. These results show for the first time that MMOB exerts influence at late as well as early steps in the catalytic cycle. They also suggest that MMOB plays a critical role in determining the ability of MMO to distinguish between methane and larger substrates.


Assuntos
Mutagênese Sítio-Dirigida , Oxigenases/genética , Oxigenases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Catálise , Dietil Pirocarbonato/química , Ativação Enzimática/genética , Furanos/química , Histidina/genética , Cinética , Metano/química , Methylosinus trichosporium/enzimologia , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Dados de Sequência Molecular , Oxigenases/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genética
7.
J Biol Chem ; 276(3): 1945-53, 2001 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-11056161

RESUMO

Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O2, and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the alpha3beta3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe2+ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O2. However, upon reduction of the Rieske cluster and exposure to naphthalene and O2, approximately 0.85 cis-diol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.


Assuntos
Complexos Multienzimáticos/metabolismo , Oxigênio/metabolismo , Oxigenases/metabolismo , Catálise , Dioxigenases , Espectroscopia de Ressonância de Spin Eletrônica , Oxirredução
8.
Biochim Biophys Acta ; 1543(1): 47-59, 2000 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-11087940

RESUMO

The soluble methane monooxygenase (MMO) system isolated from Methylosinus trichosporium OB3b catalyzes the adventitious oxidation of alkyl substituted methylcyclopropanes. If the chemical mechanism of C-H activation by MMO involves formation of a radical or carbocation intermediate at the methyl C-H of these 'radical clock' substrates, then cyclopropyl ring opened alcohols may appear in the product mixture due to rearrangement of the intermediate. The lifetime of radical intermediates can be determined from known rearrangement rate constants, k(r). Rearrangement was observed during the oxidation of 1,1,2,2-tetramethylcyclopropane (k(r)=1.7-17. 5x10(8) s(-1), 30 degrees C) but not for cis- or trans-1, 2-dimethylcyclopropane (k(r)=1.2-6.4x10(8) s(-1), 30 degrees C) or the very fast radical clock, trans-2-phenylmethylcyclopropane (k(r)=3.4x10(11) s(-1), 30 degrees C). The results show that the occurrence of rearranged products fails to correlate with either the chemical nature of the C-H bond being broken, which is very similar for all of the methylcyclopropanes studied here, or the magnitude of the radical k(r) value. This study suggests that the steric properties of the substrate play an important role in determining the outcome of the reaction. Substrates with bulky substituents near the C-H bond that is attacked appear to yield intermediates with sufficient lifetimes to rearrange. In contrast, substrates with less steric bulk are postulated to be able to approach the reactive oxygen species in the MMO active site more closely so that intermediates are either rapidly quenched or undergo subsequent interaction with the dinuclear iron cluster of MMO that prevents rearrangement.


Assuntos
Ciclopropanos/química , Methylosinus trichosporium/enzimologia , Oxigenases/química , Radicais Livres , Cinética , Modelos Químicos , Oxirredução , Oxigenases/isolamento & purificação , Estereoisomerismo , Especificidade por Substrato , Temperatura
9.
Biochemistry ; 39(44): 13503-15, 2000 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-11063587

RESUMO

The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.


Assuntos
Oxigenases/química , Catálise , Deutério/química , Ativação Enzimática , Ferro/química , Cinética , Metaloproteínas/química , Metano/química , Methylosinus trichosporium/enzimologia , Modelos Químicos , Oxirredução , Espectrofotometria , Especificidade por Substrato , Temperatura , Termodinâmica
10.
Appl Environ Microbiol ; 66(9): 4119-23, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10966438

RESUMO

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.


Assuntos
Proteínas de Arabidopsis , Dioxigenases , Peróxido de Hidrogênio/farmacologia , Oxigenases/metabolismo , Pseudomonas putida/enzimologia , Aerobiose , Catecol 2,3-Dioxigenase , Genes Reporter , Cinesinas/genética , Cinesinas/metabolismo , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas putida/genética , Pseudomonas putida/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/metabolismo
12.
Proc Natl Acad Sci U S A ; 96(14): 7905-9, 1999 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-10393920

RESUMO

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.


Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Etilenos/biossíntese , Ferro/metabolismo , Reguladores de Crescimento de Plantas/biossíntese , Aminoácido Oxirredutases/isolamento & purificação , Clonagem Molecular , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli , Cinética , Modelos Químicos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
13.
Biochemistry ; 38(19): 6178-86, 1999 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-10320346

RESUMO

The soluble form of methane monooxygenase (MMO) isolated from methanotrophic bacteria catalyzes the O2-dependent conversion of methane to methanol, as well as the adventitious oxidation of many other hydrocarbons. In past studies, it was reported that the oxidation reaction of methylcubane, a radical clock substrate, catalyzed by MMO from Methylococcus capsulatus (Bath) gave only cubylmethanol as the product rather than methylcubanol(s) or rearranged products characteristic of a radical formed on the methyl group [Choi, S.-Y., Eaton, P. E., Hollenberg, P. F., Liu, K. E., Lippard, S. J., Newcomb, M., Putt, D. A., Upadhyaya, S. P., and Xiong, Y. (1996) J. Am. Chem. Soc. 118, 6547-6555]. Such a substrate radical intermediate would be expected if the mechanism of MMO involves hydrogen atom abstraction as indicated by many previous mechanistic studies. Here it is shown that the reaction of methylcubane with the reconstituted MMO system from Methylosinus trichosporium OB3b yields both cubylmethanol and methylcubanols, with methyl hydroxylation favored over cubyl hydroxylation. This unexpected regioselectivity indicates steric effects on the reaction in agreement with past product distribution studies. In addition, the apparent majority product of the reaction is tentatively assigned as one of the possible rearranged products for this radical probe, on the basis of gas chromatography and mass spectrometry data. This result suggests the formation of a radical intermediate in the reaction, thus supporting a radical-based mechanism for this form of MMO.


Assuntos
Bactérias/enzimologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Oxigenases/metabolismo , Sítios de Ligação , Hidroxilação , Oxirredução , Especificidade por Substrato
14.
Biochemistry ; 38(18): 5799-812, 1999 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-10231531

RESUMO

Methane monooxygenase (MMO) is a nonheme iron-containing enzyme which consists of three protein components: a hydroxylase (MMOH), an NADH-linked reductase (MMOR), and a small "B" component (MMOB) which plays a regulatory role. Here, 1H, 13C, 15N heteronuclear 2D and 3D NMR spectroscopy has been used to derive the solution structure of the 138 amino acid MMOB protein in the monomer state. Pulse field gradient NMR self-diffusion measurements indicate predominant formation of dimers at 1 mM MMOB and monomers at or below 0.2 mM. MMOB is active as a monomer. Aggregate exchange broadening and limited solubility dictated that multidimensional heteronuclear NMR experiments had to be performed at a protein concentration of 0.2 mM. Using 1340 experimental constraints (1182 NOEs, 98 dihedrals, and 60 hydrogen bonding) within the well-folded part of the protein (residues 36-126), MMOB structural modeling produced a well-defined, compact alpha/beta fold which consists of three alpha-helices and six antiparallel beta-strands arranged in two domains: a betaalphabetabeta and a betaalphaalphabetabeta. Excluding the ill-defined N- and C-terminal segments (residues 1-35 and 127-138), RMS deviations are 1.1 A for backbone atoms and 1.6 A for all non-hydrogen atoms. Compared to the lower resolution NMR structure for the homologous protein P2 from the Pseudomonas sp. CF600 phenol hydroxylase system (RMSD = 2.48 A for backbone atoms) (Qian, H., Edlund, U., Powlowski, J., Shingler, V., and Sethson, I. (1997) Biochemistry, 36, 495-504), that of MMOB reveals a considerably more compact protein. In particular, MMOB lacks the large "doughnut" shaped cavity reported for the P2 protein. This difference may result from the limited number of long-range NOEs that were available for use in the modeling of the P2 structure. This NMR-derived structure of MMOB, therefore, presents the first high-resolution structure of a small protein effector of a nonheme oxygenase system.


Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxigenases/química , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Methanococcaceae/enzimologia , Dados de Sequência Molecular , Oxigenases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soluções , Relação Estrutura-Atividade , Especificidade por Substrato
15.
Biochemistry ; 38(14): 4423-32, 1999 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-10194363

RESUMO

The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (kobs = 22 +/- 2 s-1 at pH 7.7; kobs = 26 +/- 2 s-1 at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kP = 9.1 +/- 0.9 s-1 achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kQ = 2.6 +/- 0.1 s-1 achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of kH/kD = 1.3 +/- 0.1 (P formation) and kH/kD = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.


Assuntos
Methylococcaceae/enzimologia , Oxigênio/química , Oxigenases/química , Catálise , Deutério/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Computação Matemática , Modelos Químicos , Prótons , Solventes
16.
Biochemistry ; 37(8): 2131-44, 1998 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9485360

RESUMO

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.


Assuntos
Protocatecoate-3,4-Dioxigenase/química , Protocatecoate-3,4-Dioxigenase/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Eletroquímica , Ferro/química , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Mutação Puntual , Conformação Proteica , Proteus mirabilis/genética , Protocatecoate-3,4-Dioxigenase/genética , Pseudomonas fluorescens/enzimologia , Pseudomonas fluorescens/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tirosina/química
17.
Biochemistry ; 36(46): 14044-55, 1997 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-9369476

RESUMO

EPR-silent, chemically reduced protocatechuate 3,4-dioxygenase (Er) binds NO at the active site Fe2+ to yield an EPR-active, S = 3/2 species that blocks subsequent binding of all other exogenous ligands. In contrast, addition of NO to a preformed Er.CN- complex yields an EPR-active, S = 1/2 species [Er.(CN)x.NO] that exhibits resolved superhyperfine splitting from 13CN-, 15/14NO, and a protein-derived 14N. Simulations of the EPR spectra observed for the Er.(CN)x.NO complex formed with 12CN- and 13CN- (1:1) show that CN- binds in two iron ligand sites (x >/= 2). The two cyanides exhibit similar, but distinguishable, hyperfine coupling constants. This demonstrates unambiguously that at least three exogenous ligands (two cyanides and NO) can bind to the Fe2+ simultaneously and strongly suggests that at least one histidine ligand is retained in the complex. The Er.(CN)>/=2.NO complex readily exchanges both of the bound cyanides for the substrate analog, 2-hydroxyisonicotinic acid N-oxide (INO), to form a Er.INO.NO complex exhibiting the same S = 3/2 type EPR spectrum that is observed for this complex in the absence of CN-. Because the dead-end Er.NO complex does not accumulate during the exchange, the results suggest that Er.(CN)>/=2. NO and Er.INO.NO are in conformational states that allow facile exchange of INO and CN- but not NO. The results are interpreted in the context of the known X-ray crystal structures for the ferric form of the resting enzyme (Eox) and numerous Eox.substrate, inhibitor, and CN- complexes, all of which have a charge neutral iron center. It is proposed that the binding of one CN- causes dissociation of an anionic endogenous ligand which begins a series of conformational changes analogous to those initiated by anionic substrate binding to Eox. This results in a new unique coordination site for NO, and a new second site for CN-; both cyanide sites are utilized when the enzyme subsequently binds substrates or INO.


Assuntos
Cianetos/metabolismo , Óxido Nítrico/metabolismo , Protocatecoate-3,4-Dioxigenase/metabolismo , Cianetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Ferro , Ligantes , Modelos Químicos , Óxido Nítrico/química , Oxirredução , Protocatecoate-3,4-Dioxigenase/química
18.
Biochemistry ; 36(38): 11504-13, 1997 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-9298971

RESUMO

The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA. Fe(III) chelate complex. 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond. This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta). This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/química , Compostos Férricos/química , Protocatecoate-3,4-Dioxigenase/química , Anaerobiose , Brevibacterium/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Pseudomonas putida/enzimologia , Especificidade da Espécie , Análise Espectral Raman
19.
Biochemistry ; 36(33): 10039-51, 1997 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-9254599

RESUMO

Protocatechuate 3,4-dioxygenase (3,4-PCD) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate to produce beta-carboxy-cis, cis-muconate. Crystal structures of Pseudomonas putida3,4-PCD [quaternary structure of (alphabetaFe3+)12] complexed with seven competitive inhibitors [3-hydroxyphenylacetate (MHP), 4-hydroxyphenylacetate (PHP), 3-hydroxybenzoate (MHB), 4-hydroxybenzoate (PHB), 3-fluoro-4-hydroxybenzoate (FHB), 3-chloro-4-hydroxybenzoate (CHB), and 3-iodo-4-hydroxybenzoate (IHB)] are reported at 2.0-2.2 A resolution with R-factors of 0. 0.159-0.179. The inhibitors bind in a narrow active site crevasse lined with residues that provide a microenvironment that closely matches the chemical characteristics of the inhibitors. This results in as little as 20% solvent-exposed surface area for the higher-affinity inhibitors (PHB, CHB, and FHB). In uncomplexed 3,4-PCD, the active site Fe3+ is bound at the bottom of the active site crevasse by four endogenous ligands and a solvent molecule (Wat827). The orientations of the endogenous ligands are relatively unperturbed in each inhibitor complex, but the inhibitors themselves bind to or near the iron in a range of positions, all of which perturb the position of Wat827. The three lowest-affinity inhibitors (MHP, PHP, and IHB) yield distorted trigonal bipyramidal iron coordination geometry in which the inhibitor C4-phenolate group displaces the solvent ligand. MHB binds within the active site, but neither its C3-OH group nor the solvent molecule binds to the iron. The C4-phenolate group of the three highest-affinity inhibitors (PHB, CHB, and FHB) coordinates the Fe3+ adjacent to Wat827, resulting in a shift in its position to yield a six-coordinate distorted octahedral geometry. The range of inhibitor orientations may mimic the mechanistically significant stages of substrate binding to 3, 4-PCD. The structure of the final substrate complex is reported in the following paper [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066].


Assuntos
Inibidores Enzimáticos/química , Protocatecoate-3,4-Dioxigenase/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Simulação por Computador , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Cinética , Ligantes , Dados de Sequência Molecular , Estrutura Molecular , Protocatecoate-3,4-Dioxigenase/química , Pseudomonas putida/enzimologia
20.
Biochemistry ; 36(33): 10052-66, 1997 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-9254600

RESUMO

Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes a ferric ion to catalyze the aromatic ring cleavage of 3,4-dihydroxybenzoate (PCA) by incorporation of both atoms of dioxygen to yield beta-carboxy-cis, cis-muconate. The crystal structures of the anaerobic 3,4-PCD.PCA complex, aerobic complexes with two heterocyclic PCA analogs, 2-hydroxyisonicotinic acid N-oxide (INO) and 6-hydroxynicotinic acid N-oxide (NNO), and ternary complexes of 3,4-PCD.INO.CN and 3,4-PCD. NNO.CN have been determined at 2.1-2.2 A resolution and refined to R-factors between 0.165 and 0.184. PCA, INO, and NNO form very similar, asymmetrically chelated complexes with the active site Fe3+ that result in dissociation of the endogenous axial tyrosinate Fe3+ ligand, Tyr447 (147beta). After its release from the iron, Tyr447 is stabilized by hydrogen bonding to Tyr16 (16alpha) and Asp413 (113beta) and forms the top of a small cavity adjacent to the C3-C4 bond of PCA. The equatorial Fe3+ coordination site within this cavity is unoccupied in the anaerobic 3,4-PCD.PCA complex but coordinates a solvent molecule in the 3,4-PCD.INO and 3,4-PCD.NNO complexes and CN- in the 3,4-PCD.INO.CN and 3,4-PCD.NNO.CN complexes. This shows that an O2 analog can occupy the cavity and suggests that electrophilic O2 attack on PCA is initiated from this site. Both the dissociation of the endogenous Tyr447 and the expansion of the iron coordination sphere are novel features of the 3,4-PCD. substrate complex which appear to play essential roles in the activation of substrate for O2 attack. Together, the structures presented here and in the preceding paper [Orville, A. M., Elango, N. , Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10039-10051] provide atomic models for several steps in the reaction cycle of 3,4-PCD and related Fe3+-containing dioxygenases.


Assuntos
Compostos Férricos/química , Protocatecoate-3,4-Dioxigenase/química , Cristalografia por Raios X , Ligantes , Estrutura Molecular , Protocatecoate-3,4-Dioxigenase/metabolismo , Especificidade por Substrato
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