Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.

Substrate-modulated reactions of putidamonooxin. The nature of the active oxygen species formed and its reaction mechanism.

1. 4-Methoxybenzoate monooxygenase is fairly nonspecific. The enzyme system with putidamonooxin as its oxygen-activating component catalyses: (a) O-, 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, [FeO2]+, 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 H2O2. 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, [FeO2]+, 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.

Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida.

The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components. As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM. Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase. The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.

Dioxygen-activating iron center in putidamonooxin. Electron spin resonance investigation of the nitrosylated putidamonooxin.

The mononuclear non-haem iron center is the dioxygen-binding site of putidamonooxin which is the dioxygen-activating component of the 4-methoxybenzoate monooxygenase. Replacement of dioxygen by nitrosyl leads to the formation of a rather stable Fe3+ X NO- complex which is characterized by electron spin resonance (ESR) at g approximately equal to 4 and g approximately equal to 2. The ESR features can be composed by two spectral components which are characterized by different tetragonal distortions of the axial symmetry. Binding of 4-hydroxybenzoate, which is the product of the enzymatic reaction, leads to the formation of an ESR spectrum with pure axial symmetry. After binding of 4-methoxybenzoate, i.e. the physiological substrate of the monooxygenase, only one spectral component, i.e. that with a small tetragonal distortion, is observed. Binding of substrate analogues, like 4-aminobenzoate and 4-trifluoromethylbenzoate, leads to a spectral heterogeneity with variable amounts of the ESR component with a large tetragonal distortion. Benzoate induces an ESR spectrum with only that spectral component with large tetragonal distortion. The iron-depleted substrate-free form of the enzyme, ligated with NO, also shows ESR heterogeneity, i.e. both spectral components overlap, with 60% of the component with large tetragonal distortion. Binding of 4-methoxybenzoate leads to the occurrence of a pure spectrum, i.e. with small tetragonal distortion, whereas binding of benzoate leads to a pure spectrum with large tetragonal distortion. Thus, the structural heterogeneity is removed by binding of both the ligand (NO) and substrate. The Fe3+ X NO- complex is discussed as an analogue of the native oxy complex Fe3+ X O2-.

Mössbauer investigation of the cofactor iron of putidamonooxin.

Mononuclear non-heme cofactor iron of putidamonooxin has been investigated in the binary oxidized 'enzyme X substrate' complex and in the ternary 'enzyme X substrate X NO' complex via Mössbauer spectroscopy. The experimental spectra were analyzed on the basis of the spin-Hamiltonian formalism. The resulting fine and hyperfine structure parameters are compared with literature values of similar compounds. From this comparison we conclude that in the binary complex (reduced and oxidized) the mononuclear non-heme cofactor iron has a coordination number higher than four. Additionally, the cofactor iron shows remarkable spectral similarities with iron in protocatechuate 3,4-dioxygenase, though the catalytic properties of the iron sites in the two proteins are different. The data obtained form the ternary 'enzyme X substrate X NO' complex indicate that the cofactor iron (a) is in the ferric intermediate spin state (S = 3/2) and (b) is pentacoordinated, which means that upon NO binding to the reduced cofactor iron at least one ligand has to be released. Comparing our data with literature values suggests that the cofactor iron in the binary as well as in the ternary NO complex is not directly bound to a sulfur atom, though biochemical arguments seem to indicate the opposite.

Mössbauer studies on the active Fe ... [2Fe-2S] site of putidamonooxin, its electron transport and dioxygen activation mechanism.

Putidamonooxin, the oxygenase of a 4-methoxybenzoate monooxygenase enzyme system, catalyzes the oxidative O-demethylation of the substrate 4-methoxybenzoate in conjunction with the NADH:putidamonooxin oxidoreductase. Putidamonooxin is a conjugated iron-sulfur protein which needs iron ions as cofactors for its enzymatic activity. Putiamonooxin was isolated from Pseudomonas putida, which was grown on a 57Fe-enriched culture medium. Thus putidamonooxin was enriched in vivo with 57Fe up to about 80%. During our Mössbauer study of putidamonooxin a number of parameters have been varied: (a) the oxidation state of putidamonooxin (oxidized, reduced and aerobically reoxidized); (b) the substrate bound to putidamonooxin (4-methoxybenzoate, benzoate, 4-tert-butylbenzoate); (c) the temperature between 2.7 K and 245 K; (d) the applied magnetic field between 0 and 0.1 T and (e) the amount of iron cofactor. From our Mössbauer results it is obvious that the iron-sulfur centers of putidamonooxin are [2 Fe-2S] clusters similar to those of the plant-type ferredoxins. Further, we have evidence for the existence of iron ions (one per [2 Fe-2S] cluster), which serve as cofactors for the dioxygen activation, functioning as the dioxygen binding site and mediating the electron flow from the [2 Fe-2S] cluster to dioxygen.

Dioxygen activation by putidamonooxin. The oxygen species formed and released under uncoupling conditions.

In the presence of substrates not favourable for hydroxylation, more than 80% of the dioxygen consumed by purified, reconstituted 4-methoxybenzoate monooxygenase appears in the reaction mixture as hydrogen peroxide. We have investigated whether under these conditions (a) reduced putidamonooxin, the oxygenase of this enzyme system, either autoxidizes in the presence of dioxygen, with liberation of superoxide anion radicals which then disproportionate to H2O2 and O2, or (b) dioxygen is reduced by two sequential single-electron steps leading to the active oxygen species that forms hydrogen peroxide directly when inactivated by protonation. Quantitative estimation of O-2 radicals, with either succinylated ferricytochrome c or epinephrine used as O-2 scavengers, revealed that only about 6% of the total electron flux channelled via putidamonooxin to dioxygen led to the monovalent reduction on dioxygen. This means that not more than 3% of the hydrogen peroxide found under uncoupling conditions arises from the rapid bimolecular disproportionation of initially formed O-2 radicals. Inconsistent results were obtained when lactoperoxidase was used as an O-2 trap. Our measurements indicate that the conversion of lactoperoxidase into compound III is an inappropriate method of detecting any O-2 radicals that may be found by the uncoupled 4-methoxybenzoate monooxygenase. The stoichiometry of about 1:1 for O2 uptake: H2O2 formation indicates that under uncoupling conditions H2O is virtually not formed. The role of [FeO2]+ as the active oxygenating species of putidamonooxin is discussed.

Chemical and spectral properties of putidamonooxin, the iron-containing and acid-labile-sulfur-containing monooxygenase of a 4-methoxybenzoate O-demethylase from Pseudomonas putida.

Gel chromatography indicates that putidamonooxin has a molecular weight of about 126,000. On the other hand, the amino acid composition and the iron-to-protein ratio point to a minimal molecular weight of 33,000 and 31,000 respectively. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme migrated as a homogeneous band corresponding to a molecular weight of about 40,000. The number of spots found in the tryptic peptide map of the carboxymethylated and digested enzyme indicates that putidamonooxin is composed of three or four identical subunits. After covalent cross-linking of the subunits with dimethyl suberimidate and subsequent dodecylsulfate electrophoresis the main bands were in the molecular weight range of 40,000, 87,000 and 124,000. These findings lead us to propose that putidamonooxin is either a trimer or tetramer. The amino acid composition of putidamonooxin and related data calculated from this are given. The isoelectric point was shown by isoelectric focusing to be a pH 4.7. Low-temperature optical spectra of the reduced and oxidized enzyme as well as of three different putidamonooxin.substrate complexes are given together with those recorded at 10 degrees C. Enzyme.substrate binding spectra are observed with the oxidized putidamonooxin but not with the reduced enzyme. For the oxidized putidamonooxin a molar absorption coefficient at 455nm of 14.7mM-1 cm-1 was determined. Ks values of putidamonooxin towards different substrates and substrate analogues (i.e. tight couplers, partial uncouplers and uncouplers) are presented and possible reasons for the difference between the Ks values here obtained and the previously reported Km values are discussed.

Kinetic studies on a 4-methoxybenzoate O-demethylase from Pseudomonas putida.

A direct, sensitive and reliable photometric assay procedure for monitoring the activity of non-specific 4-methoxybenzoate O-demethylases of microorganisms is described. The assay is based on the O-demethylation of 3-nitro-4-methoxybenzoate to the yellow-coloured product 3-nitro-4-hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4-methoxybenzoate monooxygenase (O-demethylating) and a reductase from Pseudomonas putida have been investigated. It has been found that the KM value of the monoxygenase of this enzyme system towards different substrates (i.e. tight couplers, uncouplers and partial uncouplers) rises from the extremely low value of 0.07 muM for the tight couplers to about 55 muM for the uncouplers. The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only batho-phenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system. The affinity of the reductase towards NADH was found to be approximately 200-fold greater than that towards NADPH. Futhermore, the affinity of this reductase to NADH depended on the nature of the electron acceptor. The affinity to NADH was more than 10 times higher when the monooxygenase-substrate complex was used as the electron acceptor, than when cytochrome c or 2,6-dichloroindophenol was used. These differences are discussed on the basis of enzyme-enzyme interactions between the reductase and the monooxygenase.

A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-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 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 415 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 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.