1. 4-Methoxybenzoate monooxygenase is fairly nonspecific. The enzyme system with putidamonooxin as its oxygen-activating component catalyses: (a) 0-, S-and N-demethylation; (b) the oxygenation of 4-methylbenzoate and 4-methylmercaptobenzoate, with formation of 4-carboxybenzyl alcohol and 4-carboxyphenylmethyl sulfoxide, respectively, and (c) attack of the aromatic ring of 4-and 3-hydroxybenzoate and 4-aminobenzoate, yielding 3,4-dihydroxybenzoate and 4-amino-3-hydroxybenzoate, respectively.2. Compounds which are bound by the active sites of putidamonooxin have two essential features in common: a planar aromatic ring system, and a free carboxyl group attached to it.3. By a substrate-modulated reaction putidamonooxin can be induced to function not only as a monooxygenase but also as a peroxotransferase, i.e. it incorporates both atoms of the activated oxygen molecule into a substrate molecule. This finding supports the hypothesis that a mesomeric state of the iron . peroxo complex, [FeO,] ', is indeed the active oxygenating species of putidamonooxin.4. The lifetime of the ternary complex consisting of enzyme . iron-peroxo-complex . substrate is significantly prolonged by uncoupling and partially uncoupling substrates, except when it is inactivated by protonation of the iron . peroxo complex by a proton transported into the active sites by a special kind of substrate (i.e. isomers of monoaminobenzoate), with the direct formation of H202.5. The lifetime of the active oxygen species is determined by (a) the rate of the oxygenation reaction in the presence of tight-coupling substrates and (b) the rate of the oxygenation reaction as well as detoxification by the availability of a dissociable proton in the presence of partial uncoupling (and uncoupling) substrate analogues.6. The rate of the oxygenation reaction depends on the lifetime of the active oxygen species, [Fe02J+, in the presence of partial uncoupling substrates.7. The iron . peroxo complex attacks an aromatic ring system according to the empiric rules of electrophilic substitution, whereas the attack of aliphatic substituents at the aromatic ring is controlled by steric criteria.The 4-methoxybenzoate monooxygenase from Pseudomonas putidu (DSM no. 1868), in the presence of NADH and oxygen, catalyses the conversion of its physiological substrate 4-methoxybenzoate to 4-hydroxybenzoate via an unstable semiacetal intermediate [l]. The two constituents of this enzyme system, the oxygen-activating component called putidamonooxin and its reductase, the NADH -putidamonooxin oxidoreductase [a]
A strain of Pseudomonus putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the 0-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components : an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase.The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently.The absolute spectrum of the oxidized reductase displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores.The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 41 5 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the velocity and the extent of reduction of the iron and labile-sulfur-containing monooxygenase by the NADH-dependent reductase.Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i e . , iron-containing and labile-sulfur-containing monooxygenases.From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed. The 4-methoxybenzoate 0-demethylase from Pseudomonas putidu is a fairly unspecific enzyme. In the presence of NADH and molecular oxygen, the aliphatic C-H bond as well as the aromatic ring are attacked [ll]. Studies on substrate binding and oxygen aromatic ring, rather than the side chains of the Abbreviations. CD, circular dichroism; EPR, electron paramagnetic resonance, 2, mean g-value = 1/3 (gx + g, + gz).
The interaction of substrates with a 4-methoxybenzoate 0-demethylating enzyme system was studied by use of crude cell-free extracts and also by the purified enzyme system. The two components of the enzyme system, an iron-containing flavoprotein and an iron-sulphur protein, were obtained in pure state from Pseudomoms putida grown on 4-methoxybenzoate as the sole carbon source. The purified enzyme system requires NADH and oxygen as cofactors and acts on various substrates. The highest affinity is found for 4-methoxybenzoate, but also N-methyl-4-aminobenzoate is demethylated and 4-alkylbenzoates are hydroxylated a t the side chain. The enzyme is rather specific for para-substituted benzoic acid derivatives whereas 3-methoxybenzoate and 4-hydroxy-3-methoxybenzoate are demethylated slowly. The enzyme is also able to hydroxylate the aromatic ring. This is shown by the isolation of 3,4-dihydroxybenzoate as the hydroxylation product of 3-hydroxy-or 4-hydroxy-benzoate, respectively. Studies on substrate binding and oxygen consumption with substrate analogues showed an absolute requirement for the carboxy group a t the aromatic ring. Benzoic acid derivatives without a suitable CH-bond uncouple oxygen uptake with a concomitant formation of hydrogen peroxide. Measurements of oxygen consumption indicate that the affinity towards oxygen is substrate dependent, probably due to steric alterations as a consequence of substrate binding.The biodegradation of aromatic 0-alkyl ethers in bacteria is generally initiated by monooxygenase systems requiring reduced pyridine nucleotides and oxygen. An activated form of oxygen attacks the &-carbon atom and the methyl group is oxidatively eliminated probably via an unstable semiacetalUnlike the well-known nonspecific 0-dealkylating system in mammalian liver microsomes [2,3], the bacterial monooxygenases have been found to exhibit rather high specificity for the phenolic ethers on which the bacteria were grown.Cartwright et al. [4-61 have reported the existence of 3-and 4-methoxybenzoate demethylases in cell extracts from Pseudomonas fluorescens and Pseudomonas strain P 6. These enzymes again are different by pH-optima, sensitivity to inhibitors and stability against dialysis from the 4-methoxybenzoate 0-demethylating system which we have isolated from Pseudomonas putida [ 11. Recent investigations by Ribbons [7,8] on 3-methoxybenzoic acid 0-demethylases from P. aeruginosa and P. testosteroni in agreement with the results of Cartwright and our findings indicate that these monooxygenases may be multienzyme systems with similar structures.We have highly purified and characterized an iron-containing flavoprotein and an iron-sulphur protein (EPR at g = 1.91) as components of the 0-demethylating enzyme system from P. putidu grown on 4-methoxybenzoate [9]. There is no indication for the participation of a cytochrome, although Cartwight et al. [iO,ii] have recently described cytochrome P450 and another heme component in 0-demethylase systems from Noeardia NH 1 and P. fluorescens Tp, respectively. ...
4-Methoxybenzoate monooxygenase isolated from a species of Pseudomonas putida can be separated in two components, NADH-putidamonooxin oxidoreductase and putidamonooxin, the latter contains the dioxygenactivating site. Binding of tight coupling substrates leads to monooxygenase activity of this enzyme system with substrate hydroxylation, whereas binding of uncoupling substrates leads to oxidase activity with production The NADH-putidamonooxin oxidoreductase when reduced with NADH exhibits an electron spin resonance (ESR) spectrum with rhombic symmetry and an isotropic resonance at g = 2.0033. The average g value of 1.96 and the temperature behaviour of the rhombic spectrum are indicative for (2Fe-2s) clusters. In the range of pH 6.8-8.2 the isotropic ESR signal attributed to FMN shows the characteristic linewidth of the blue semiquinone form.The reduced putidamonooxin shows an ESR spectrum with rhombic symmetry and an average g value of 1.89 caused by a pronounced g anisotropy. An identical g anisotropy is observed for the Rieske-cluster.The "Fe-enriched (2Fe-2s) clusters show hyperfine interaction between the electron spin and the nuclear spin of the iron atoms in two principal directions of the A-tensor, the resonance in the third direction is not resolved. For one direction equivalent hyperfine constants with a = 1.23 mT are observed, whereas for the other direction the hyperfine constants are inequivalent with a = 2.0 mT and a < 0.05 mT respectively indicating different spin densities at the iron atoms in the cluster. Hyperfine constants and g tensor of the reduced cluster are not influenced by binding of substrate to putidamonooxin.The ESR feature in the range of g = 9.6 -4.3 is characteristic for a mononuclear non-heme iron center in oxidized putidamonooxin. The temperature behaviour of the resonance at g5 = 4.293 in the range of 3.6-80 K indicates a zero-field splitting with D = 0.9 0.1 cm-'. Therefore, we assume that this iron center is probably coordinated by oxygen and nitrogen atoms. On the basis of g values this mononuclear non-heme iron center has at least two different microenvironments generating rhombic ( E / D = 0.33) and tetragonal symmetry (E/D = 0.05 -0.15). Whereas the iron with tetragonal symmetry is sensitive to substrate-binding, the iron with rhombic symmetry seems to be independent of substrate-binding. Nevertheless, both symmetry species are involved in the activation of dioxygen as the iron-sulfur clusters can only be aerobically oxidized if mononuclear iron centers are present in a 1 : 1 ratio. Substitution of the mononuclear non-heme iron center by 57Fe leads to a line-broadening of the resonance at g5 = 4.293 indicating a splitting constant of 0.8 5 0.2 mT.It is excluded, by comparing aerobically and anaerobically oxidized putidamonooxin, that the iron center with tetragonal symmetry belongs to a ternary dioxygen ' enzyme . substrate complex. of H202.
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