Uncoupled Oxidation Research Articles

Changing the Substrate Reactivity of 2-Hydroxybiphenyl 3-Monooxygenase from Pseudomonas azelaica HBP1 by Directed Evolution

The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased V(max) from 0.22 to 0.43 units mg(-1) protein and decreased the K'(m) from 588 to 143 microm, improving k'(cat)/K'(m) by a factor of 8.2. With 2-tert-butylphenol as the substrate, k'(cat) was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K'(m) for guaiacol decreased with this mutant, but the K'(m) for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1% sequence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile(244) of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val(368) and Leu(417) are not close to the active site and would not have been obvious candidates for modification by rational design.

Magnetic Resonance Spectroscopic Studies of Neurotransmitters and Energy Metabolism in Epilepsy

Summary: The application of magnetic resonance spectroscopy (MRS) to study brain glutamate and γ‐aminobutyric acid (GABA) metabolism and the coupling of neurotransmitter cycling to neuroenergetics has provided several new, and controversial, insights into the relationship of brain metabolism and function in humans and animal models. Contrary to the previous view of separate metabolic and neurotransmitter pools of glutamate, the glutamate–glutamine cycle has been shown to be a major metabolic pathway tightly coupled to cerebral electrical activity and glucose metabolism. The glycogen‐shunt model provides a mechanistic explanation for the uncoupling of glucose consumption and oxidation during enhanced cerebral electrical activity. A ketogenic diet improves the cerebral energy state of patients with refractory epilepsy. Ketones may act by providing oxidative fuel for non–neurotransmission‐linked cerebral energy needs. This resulting sparing effect on glucose essentially provides an extra source of substrate to eliminate synaptic glutamate, which ultimately reduces excitability. Postictal glutamate reuptake is threefold slower in the gliotic yet epileptogenic human hippocampus, suggesting partial dysfunction of the glia, slowing the glutamate–glutamine cycle. Glutamate and GABA metabolism are coupled at the metabolic and functional levels. GABAergic neurons are dependent on glia to provide glutamine for GABA synthesis (GABA–glutamine cycle). Occipital GABA levels are below normal in many patients with refractory complex partial seizures and juvenile myoclonic epilepsy. Patients with lower GABA levels have slower rates of GABA synthesis. In the human visual cortex, GABA concentrations, and therefore rates of GABA synthesis, appear to be coupled to functional activity and inversely related to cortical excitability. Studies of glutamate/GABA/glutamine cycles, ketone body metabolism, and functional neuroenergetics should provide further insight into the metabolic mechanisms controlling cerebral excitability.

Involvement of Reactive Oxygen Species in Plant Mitochondrial Response to Low-Temperature Stress

The review presents data for the generation of the reactive oxygen species (ROS) in mitochondria during low-temperature stress. The relation of lipid peroxidation processes in membranes to the induction of low-temperature stress is demonstrated. The data for the involvement of the mitochondrial uncoupling proteins and the processes of uncoupling oxidation and phosphorylation in the regulation of mitochondrial ROS formation during low-temperature stress are considered.

Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling.

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.

Mildly oxidized glyceraldehyde-3-phosphate dehydrogenase as a possible regulator of glycolysis.

Influence of H2O2 on glycolysis was investigated. A hypothesis previously formulated was tested according to which a mild oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) results in uncoupling of oxidation and phosphorylation at this step of glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation of a mixture of purified glycolytic enzymes, as well as a muscle extract, in the presence of 10-100 microM H2O2 was shown to result in an increase in the rate of glycolysis. The level of lactate accumulation in the oxidized samples increased by 80-150% compared to the samples containing mercaptoethanol. No ATP was formed by the H2O2-stimulated glycolysis. Thus, H2O2 really caused uncoupling of oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in regulation of glycolysis is discussed.

AN INFLUENCE OF ANTISERUM AGAINST WINTER WHEAT STRESS UNCOUPLING PROTEIN, CSP 310, ON ENERGETIC ACTIVITY OF SOME PLANT SPECIES MITOCHONDRIA

It is determined that addition of an anti-CSP 310 antiserum to isolated mitochondria of cereals (winter rye, winter wheat, and maize) caused an increasing of mitochondrial respiratory control. In a similar manner, addition of this antiserum to isolated pea mitochondria did not cause this effect. It is shown that coupling effect of antiserum is not dependent upon the presence of bovine serum albumin in mitochondria incubation media. Therefore, these results show that the mechanism of oxidation and phosphorylation uncoupling, and participation of immunochemically related CSP 310 proteins that can be precipitated by anti-CSP 310 antiserum is specific for cereals.

Neuronal Nitric-oxide Synthase Mutant (Ser-1412 → Asp) Demonstrates Surprising Connections between Heme Reduction, NO Complex Formation, and Catalysis

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.

Mechanistic studies on prolyl-4-hydroxylase: the vitamin c requiring uncoupled oxidation

A deuteriated substrate for the human type I prolyl-4-hydroxylase was synthesized and its V/K deuterium isotope effect was determined to be 3.4 ± 0.2. This isotope effect was attributed to the uncoupled oxidation. A dehydroproline containing tetrapeptide was also found to stimulate the uncoupled oxidation.

Interaction of Endothelial and Neuronal Nitric-oxide Synthases with the Bradykinin B2 Receptor: BINDING OF AN INHIBITORY PEPTIDE TO THE OXYGENASE DOMAIN BLOCKS UNCOUPLED NADPH OXIDATION

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.

Time-courses of Growth Inhibition and Recovery for Narcotic Chemicals and 2,6-Dinitrophenol in Pollen Suspensions of Nicotiana sylvestris.

In a previous structure-activity analysis of chlorophenol and nitrophenol toxicity, the pollen tube growth test was shown to discriminate between oxidative uncoupling and narcotic mechanisms of action. To examine the suitability of the use of pollen tubes in screening for narcotic chemicals, we used tobacco pollen suspensions and performed time-course experiments on pollen tube growth inhibition and recovery after exposure to 1-butanol, 2-chloroaniline, 2,4-dichlorophenol and 2,6-dinitrophenol, during pollen culture for 22 hours. After exposure to the chemicals for 2 hours, pollen tubes exposed to 1-butanol and 2,6-dinitrophenol were able to recover, whereas recovery was poor after exposure to 2-chloroaniline and 2,4-dichlorophenol. Dilution experiments to remove the narcotics from the pollen suspension indicated that 2-chloroaniline and 2,4-dichlorophenol accumulated in the pollen grain wall, presumably due to their high octanol/water partition coefficients. Therefore, we suggest that the pollen tube growth test is not suitable for correctly predicting the narcotic potencies of highly lipophilic compounds. In the presence of 1-butanol, pollen grains did not germinate, but became characteristically enlarged. This observation suggests that 1-butanol inhibits the establishment of the cell polarity necessary for initiating pollen tube outgrowth.

Pyruvate and hydroxycitrate/carnitine may synergize to promote reverse electron transport in hepatocyte mitochondria, effectively ‘uncoupling’ the oxidation of fatty acids

In a recent pilot study, joint administration of pyruvate, hydroxycitrate (HCA), and carnitine to obese subjects was associated with a remarkable rate of body-fat loss and thermogenesis, strongly suggestive of uncoupled fatty-acid oxidation. Hepatocytes possess an uncoupling mechanism – reverse electron transport – that enables fasting ketogenesis to proceed independent of respiratory control. Electrons entering the respiratory chain at the coenzyme Q (CoQ) level via FAD-dependent acyl coA dehydrogenase, can be driven ‘up’ the chain by the electrochemical proton gradient to reduce NAD+; if these electrons are then shuttled to the cytoplasm, returning to the respiratory chain at the CoQ level, the net result is heat generation at the expense of the proton gradient, enabling the uncoupled flow of electrons to oxygen. Pyruvate's bariatric utility may stem from its ability to catalyze the rapid transport of high-energy electrons from mitochondria to the cytoplasm, thus stimulating electron shuttle mechanisms. By enabling rapid mitochondrial uptake of fatty acids and thus disinhibiting hepatocyte ketogenesis, HCA/carnitine should initiate reverse electron transport: concurrent amplification of electron shuttle mechanisms by pyruvate can be expected to accelerate this reverse electron transport, thereby decreasing the electrochemical proton gradient. As a result, hepatocytes may be able to convert fatty acids to CO2and heat with little net generation of ATP. These considerations suggest that it may be feasible to render hepatocytes functionally equivalent to activated brown fat, such that stored fat can be selectively oxidized in the absence of caloric restriction. Other measures which enhance the efficiency of hepatocyte electron shuttle mechanisms may increase the efficacy of this strategy.

Roles of key active-site residues in flavocytochrome P450 BM3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

Roles of key active-site residues in flavocytochrome P450 BM3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

Anion carriers in fatty acid-mediated physiological uncoupling.

Physiological aspects of uncoupling of oxidation and phosphorylation are reviewed in the context of involvement of mitochondrial anion carriers. It is assumed that the carriers facilitate electrophoretic translation of fatty acid anion, RCOO-, from the inner to the outer leaflet of the mitochondrial membrane, whereas back movement of the protonated fatty acid, RCOOH, from the outer to the inner leaflet represents flip-flop of RCOOH via the phospholipid bilayer of the membrane. The RCOO- transport seems to be catalyzed by the ATP/ADP and aspartate/ glutamate antiporters, dicarboxylate carrier, and uncoupling proteins (UCP1, UCP2, UCP3L, UCP3s, and plant UCP). The fatty acid uncoupling is shown to be involved in the thermoregulatory heat production in animals and plants exposed to cold, as well as in performance of respiratory functions other than ATP synthesis, i.e., formation of useful substances, decomposition of unwanted substances, and antioxidant defense. Moreover, partial uncoupling might take part in optimization of the rate of ATP synthesis in aerobic cells.

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC 50 for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.

Effects of pH on the structure and function of neuronal nitric oxide synthase.

We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.

Regulation of pyruvate dehydrogenase activity and glucose metabolism in post-ischaemic myocardium

Pyruvate dehydrogenase (PDH) is regulated both by covalent modification and through modulation of the active enzyme by metabolites. In the isolated heart, post-ischaemic inhibition of PDH, leading to uncoupling of glycolysis and glucose oxidation and a decrease in cardiac efficiency, has been described. In vivo, post-ischaemic reperfusion leads to metabolic abnormalities consistent with PDH inhibition, but the effects of ischaemia/reperfusion on PDH are not well characterized. We therefore investigated PDH regulation following transient ischaemia in vivo. In 33 open-chest dogs, the left anterior descending (LAD) was occluded for 20 min followed by 4 h reperfusion. In 17 dogs, dichloroacetate (DCA) was injected prior to reperfusion, while 16 dogs served as controls. In dogs without DCA, glucose oxidation and lactate uptake were lower in reperfused than in remote tissue, suggesting reduced flux through PDH. However, percent active and total PDH measured in myocardial biopsies were similar in both territories, excluding covalent enzyme modification or loss of functional enzyme. DCA activated PDH activity similarly in both regions and abolished differences in glucose oxidation and lactate uptake. Thus, decreased PDH flux in reperfused myocardium does not result from covalent modification or loss of total enzyme activity, but more likely from metabolite inhibition of the active enzyme. DCA leads to essentially complete activation of PDH, increases overall glucose utilization and abolishes post-ischaemic inhibition of glucose oxidation.

An uncoupling of the processes of oxidation and phosphorylation in glycolysis.

Data on alterations of the properties of glyceraldehyde-3-phosphate dehydrogenase upon oxidation of its functional groups are reviewed; a mechanism of uncoupling of oxidation and phosphorylation in glycolysis is considered. Possible ways of regulating uncoupling, and the physiological importance of this process, are discussed.

Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp.

Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated.

Purification and Characterization of 2-Hydroxybiphenyl 3-Monooxygenase, a Novel NADH-dependent, FAD-containing Aromatic Hydroxylase from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from a recombinant of Escherichia coli JM109, which efficiently expressed the hbpA gene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparent Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM for oxygen. The enzyme was inactivated by p-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the betaalphabeta-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.