Substrate‐modulated reactions of putidamonooxin

Abstract

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]

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

“…One early proposal whereby an Fe 3ϩ -(hydro)peroxo species attacks the substrate (illustrated in Scheme 3A) derived from studies on putidamonooxin. Using uncoupling substrates, which caused the production of hydrogen peroxide, in combination with solvent isotope effects, a ferric-peroxo intermediate was suggested as the species responsible for the demethylation and dihydroxylation reactions catalyzed by this enzyme (53)(54)(55)(56). More recently, this general mechanism has found support in the work of Lee (57), which showed that benzene acts as both a substrate and an uncoupler of NDO, generating H 2 O 2 during the reaction.…”

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS

“…These data indicate that it was rather unlikely that some of the substrate analogs tested uncoupled electron transfer from substrate hydroxylation. Oxygen uptake due to substrate-dependent uncoupling of electron transfer rather than true oxygenase activity has been reported for a number of oxygenases, e.g., 4-methoxybenzoate monooxygenase (10,11,67), phthalate dioxygenase (7), and salicylate hydroxylase (71).…”

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4

“…Lipscomb has proposed that the diiron-oxo clusters in methane monooxygenase are converted during catalysis to an [Fe(IV) ⅐ Fe(IV)]AO species, which would be a strong oxidant similar to the Fe(IV)AO species proposed for cytochrome P-450 (33). An iron-peroxo complex [FeO 2 ] ϩ has been proposed for the oxygenating species in 4-methoxybenzoate O-demethylase (56).…”

“…In this context, it is of interest that NDO can catalyze O-dealkylation (41), N-dealkylation, and sulfoxidation reactions (32), which are also typical cytochrome P-450 reactions. Other non-heme iron oxygenases that catalyze reactions similar to those catalyzed by cytochrome P-450 are the soluble forms of methane monooxygenase (19,33) and 4-methoxybenzoate monooxygenase (56). Ammonia monooxygenase appears to be responsible for the cytochrome P-450-type reactions catalyzed by Nitrosomonas europaea (29,54).…”