Amination of tyrosine in liver cytosol protein of male F344 rats treated with 2-nitropropane, 2-nitrobutane, 3-nitropentane, or acetoxime

Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344-351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.1-1.5 mol of 3-aminotyrosine/10(3) mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at approximately 0.01 mol/10(3) mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-O-sulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-O-sulfonates as intermediates.     

Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from 18O2 and one atom of oxygen from H218O are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.     

Synthesis and properties of 8-CN-flavin nucleotide analogs and studies with flavoproteins

A high potential analog of riboflavin with a cyano function at the 8-position was synthesized by employing novel reaction conditions, starting from 8-amino-riboflavin. This was converted to the FAD level with FAD synthetase. The reduced 8-CN-riboflavin, unlike normal reduced flavin, has a distinctive absorption spectrum with two distinctive peaks in the near ultraviolet region. The oxidation-reduction potential of the new flavin was determined to be -50 mV, approximately 160 mV more positive than that of normal riboflavin. The 8-CN-riboflavin and 8-CN-FMN were found to be photoreactive and need to be protected from exposure to light. However such complications were not encountered with protein-bound flavins. The apoproteins of flavodoxin and Old Yellow Enzyme (OYE) were reconstituted with the 8-CN-FMN and apoDAAO was reconstituted with 8-CN-FAD. Spectral properties of the enzyme-bound neutral and anionic semiquinones were determined from these reconstituted proteins. In the case of 8-CN-FMN-OYE I, it was shown that the comproportionation reaction of a mixture of reduced and oxidized enzyme bound flavin is very rapid, compared with the same reaction with native protein, resulting in approximately 100% thermodynamically stable anionic semiquinone. In the case of 8-CN-OYE I, it was shown that the rate of reduction of the enzyme bound flavin by NADPH is approximately 40 times faster, and the rate of reoxidation of reduced enzyme bound flavin by oxygen is an order of magnitude slower than with the normal FMN enzyme. This is in accord with the high oxidation-reduction potential of the flavin, which thermodynamically stabilizes the reduced enzyme.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.     

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.     

Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1

2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.     

Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum

The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet. In 2H2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.     

Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.     

Review of Rett syndrome

A FAD and [4Fe-4S]cluster-containing enzyme from Clostridium aminobutyricum catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA which involves the cleavage of an unactivated C-H bond at the beta-carbon. Transient oxidation of the substrate to an enoxy radical by FAD might facilitate the removal of this beta-proton, whereas no function could be attributed to the [4Fe-4S]cluster. In this paper the organic radical, which is formed by partial reduction of the enzyme with dithionite, was characterised as the neutral flavin semiquinone by EPR spectroscopy in H2O and D2O. The rapid electron-spin relaxation of the flavin semiquinone suggested a magnetic interaction with the [4Fe-4S]cluster. In order to obtain highly resolved information about nuclear spins in the vicinity of this paramagnetic centre, ENDOR spectroscopy was applied. The spectra were compared with those of the neutral semiquinone radicals of ferredoxin-NADP reductase and flavodoxin as well as with that of the anionic semiquinone radical of cholesterol oxidase. All ENDOR spectra showed strong couplings to the 8-methyl protons and to H-6 of the flavin. On addition of the substrates to the corresponding enzymes, the electron density changed significantly only at the 8-position. It decreased in the case of cholesterol oxidase and ferredoxin-NADP reductase, whereas an increase was observed with 4-hydroxybutyryl-CoA dehydratase. The results indicate an interaction of 4-hydroxybutyryl-CoA with the flavin as required by the proposed mechanism. Furthermore, the shift of electron density towards the benzoid ring of FAD in the dehydratase might be due to the location of the [4Fe-4S]cluster next to the 8-position as known from structurally characterised iron-sulfur flavoproteins.     

Role of tyrosine 129 in the active site of spinach glycolate oxidase

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.     

Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-corrinoid enzyme complex: stepwise reduction of iron-sulfur and corrinoid centers, the corrinoid Co2+/1+ redox midpoint potential, and overall synthesis of acetyl-CoA

Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ corrinoid at potentials existing in vivo. The reduced corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of Hexp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH2)7 CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex = 3-O-methyl-beta-D-Gal, 3-deoxy-beta-D-Gal, 3-deoxy-3-fluoro-beta-D-Gal, 3-amino-3-deoxy-beta-D-Gal, beta-D-Gul, alpha-L-Alt, or beta-L-Gal

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.     

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.     

Interaction of NADPH-adrenodoxin reductase with NADP+ as studied by pulse radiolysis

The reduction of flavin in NADH--adrenodoxin reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced directly the flavin of the reductase to form a blue semiquinone of the enzyme. Subsequently, the semiquinone decayed by dismutation to form the oxidized and fully reduced forms of the enzyme with a second-order rate constant of 4.4 x 10(4) M-1 s-1. In the presence of equimolar NADP+, the decay of eaq- accompanied an absorption increase at 400 nm, the spectrum of which, formed transiently, is identical to that of NADP radical (NADP.). Subsequently, the transient species decayed concomitantly with the formation of the semiquinone. The rate constant in the formation of the semiquinone was independent of the concentration of the enzyme (6.1 x 10(4) s-1 at pH 7.5). From these results, it is concluded that eaq- reacts with NADP+ bound to the enzyme to form NADP. initially, and subsequently, an electron flows from the NADP. to the flavin by an intracomplex electron transfer. A similar result was obtained in the reaction of CO2- or N-methylnicotinamide radical with the NADP(+)-adrenodoxin reductase complex. These results suggest that the nicotinamide moiety of NADP+ bound to the enzyme is accessible to the solvent and masks the flavin completely.     

Purification and characterization of an NADH-rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas

An NADH--rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109-115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid-point redox potentials of -295 mV and -325 mV, this FMN- and FAD-containing protein can induce the specific reduction of D. gigas rubredoxin. In contrast, rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half-inhibited by 4.2 microM p-chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.     

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.     

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.     

Catalytic properties of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki

Adenylylsulfate reductase (EC 1.8.99.2) isolated from Desulfovibrio vulgaris Miyazaki catalyzes electron transfer from dihydroflavin coenzyme (FADH2, FMNH2, or dihydroriboflavin) to adenylyl sulfate (APS), and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS. The reaction with FAD as an electron mediator was markedly stimulated in the presence of menadione. Km of the enzyme was about 0.015 mM for riboflavin and FAD in the presence of menadione. Free flavin coenzyme was found to be the normal cellular constituent. These observations suggested that free flavin coenzyme may be a physiological electron carrier for APS reductase, and the enzyme may be called AMP, sulfite:flavin oxidoreductase. Km (APS) of this enzyme is lower than 1 microM. The enzyme is not inhibited by ATP and GTP, but was inhibited by AMP and sulfite. Its extremely low Km (APS) enables this enzyme to reduce any traces of cytosolic APS which is present only at micromolar concentration, and inhibition by sulfite makes this organism utilize an energetically favorable electron acceptor, sulfite, preferentially over APS which is produced from sulfate at the cost of ATP.