Isotope effects and intermediates in the reduction of NO by P450(NOR).

The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.

Cytochromes P450 and flavin monooxygenases--targets and sources of nitric oxide.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 01 meeting in Orlando, FL. The presentations addressed the mechanisms of inhibition and regulation of cytochrome P450 and flavin monooxygenase enzymes by nitric oxide. They also highlighted the consequences of these effects on metabolism of drugs and volatile amines as well as on important physiological parameters, such as control of blood pressure, renal ion transport, and steroidogenesis. This is achieved via regulation of P450-dependent prostacyclin, hydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acid formation. Conversely, the mechanisms and relative importance of nitric oxide synthases and P450 enzymes in NO production from endogenous and synthetic substrates were also addressed.

A novel C1-using denitrifier alcaligenes sp. STC1 and its genes for copper-containing nitrite reductase and azurin.

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.

Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum.

Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.

Oxygen requirement for denitrification by the fungus Fusarium oxysporum.

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.

A positively charged cluster formed in the heme-distal pocket of cytochrome P450nor is essential for interaction with NADH.

Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gln, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br(-)) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br(-), termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system. A comparison of the predicted mechanisms between the perturbations caused by Br(-) and the mutations suggested that Arg(174) and Arg(64) play a crucial role in binding NADH to the protein. These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.

Involvement of formate as an interspecies electron carrier in a syntrophic acetate-oxidizing anaerobic microorganism in coculture with methanogens.

To determine whether formate is involved in interspecies electron transfer between substrate-oxidizing bacteria and hydrogenotrophic microorganisms under anaerobic conditions, a syntrophic acetate-oxidizing bacterium Thermacetogenium phaeum strain PB was cocultured either with a formate /H2-utilizing methanogen strain TM (designated as PB/TM coculture), or an H2-utilizing methanogen strain deltaH (designated as PB/deltaH coculture). Acetate oxidation and subsequent methanogenesis in PB/TM coculture were found to be significantly faster than in PB/deltaH coculture. Formate dehydrogenase and hydrogenase were both detected in strains PB and TM. H2 partial pressures in the PB/TM coculture were kept lower (20 to 40 Pa) than those of the PB/deltaH coculture (40 to 60 Pa) during the exponential growth phase. Formate was also detected in both PB/TM and PB/deltaH cocultures, and the concentration of formate was maintained at a lower level in the PB/TM coculture (5 to 9 microM) than in the PB/deltaH coculture. Thermodynamic calculations revealed that the concentrations of both H2 and formate severely affect the syntrophic oxidation of acetate. These results strongly indicate that not only H2 but also formate may be involved in interspecies electron transfer.

Well maintained expression of CYP genes in sandwich-culturing hepatocytes: quantitative analysis using real-time PCR method.

Sandwich-culturing is an excellent hepatocyte culturing method in drug metabolism studies, however, its advantages for gene expression of cytochrome P450 (CYP) have not been evaluated so far. The present study was undertaken to determine the utilities of sandwich-culturing hepatocytes for evaluation of CYP genes expression. Hepatocytes from male rats were cultured for 5 days between two layers of type-I collagen gel (sandwich-culturing) or over type-I collagen gel (single gel culturing). To determine the expression of CYP genes rapidly and accurately, the time course study using real-time RT-PCR quantification was conducted in the present study, and CYP2B1, CYP2B2, CYP3A2, CYP3A9 and CYP3A23 genes were measured. Albumin secretion was also measured by ELISA to evaluate cell viability. Higher expression and excellent maintenance of all CYP genes were confirmed in sandwich-culturing hepatocytes than those in single gel culturing. Particularly, significant difference in the amounts of CYP genes expression was observed between both methods after 3 days culturing. Albumin secretion was also higher in sandwich-culturing after 3 days culturing, suggesting that the cell viability of hepatocytes was maintained. These results indicate that sandwich-culturing method is much more advantageous than the ordinate method in maintaining the CYP gene expression and cell viability.

Crystal structures of cytochrome P450nor and its mutants (Ser286-->Val, Thr) in the ferric resting state at cryogenic temperature: a comparative analysis with monooxygenase cytochrome P450s.

Cytochrome P450nor (P450nor) is a heme enzyme isolated from the denitrifying fungus Fusarium oxysporum and catalyzes the NO reduction to N2O. Crystal structures of the wild type and two Ser286 mutants (Ser286-->Val, Ser286-->Thr) of P450nor have been determined for the ferric resting forms at a 1.7 A resolution at cryogenic temperature (100 K). We carried out three comparative analyses: (1) between the structures of P450nor at room temperature and cryogenic temperature, (2) between the structures of P450nor and four monooxygenase P450s, and (3) between the structures of the WT and the Ser286 mutant enzymes of P450nor. Comparison of the charge distribution on the protein surface suggests that proton and electron flow to the heme site is quite different in P450nor than in monooxygenase P450s. On the basis of the mutant structures, it was found that a special hydrogen-bonding network, Wat99-Ser286-Wat39-Asp393-solvent, acts as a proton delivery pathway in NO reduction by P450nor. In addition, the positively charged cluster located beneath the B'-helix is suggested as possible NADH binding site in P450nor, from which the direct two-electron transfer to the heme site allows to generate the characteristic intermediate in the NO reduction. These structural characteristics were not observed in structures of monooxygenase P450s, implying that these are factors determining the unique NO reduction activity of P450nor.

Fusarium oxysporum fatty-acid subterminal hydroxylase (CYP505) is a membrane-bound eukaryotic counterpart of Bacillus megaterium cytochrome P450BM3.

The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast. The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium. The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3. Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells. This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane. These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems. P450foxy was classified in the new family, CYP505.

Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium.

A novel anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium, strain PB(T), was isolated from a thermophilic (55 degrees C) anaerobic methanogenic reactor which had been treating kraft-pulp waste water. The bacterium oxidized acetate in co-culture with a thermophilic hydrogenotrophic methanogen. Strain PB(T), a gram-positive, spore-forming, rod-shaped bacterium grew optimally at 58 degrees C and pH 6.8. The bacterium grew acetogenically on several alcohols, methoxylated aromatics, pyruvate, glycine, cysteine, formate and hydrogen/CO2. Strain PB(T) also oxidized acetate with reduction of sulfate or thiosulfate as the electron acceptor. The bacterium contained MK-7 as the major quinone. The G+C content of the DNA was 53.5 mol%. Comparative 16S rDNA analysis indicated that strain PB(T) belongs to the Bacillus-Clostridium subphylum. However, it was distant from any known genera or micro-organism. The closest known relative was Thermoterrabacterium ferrireducens with 87.4% similarity. The name Thermacetogenium phaeum gen. nov., sp. nov. is proposed. The type strain is strain PBT (= DSM 12270T).

Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor.

The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous oxide (N2O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced beta-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor. These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N2O, the last step in denitrification by F. oxysporum.

Rapid reactions of peroxynitrite with heme-thiolate proteins as the basis for protection of prostacyclin synthase from inactivation by nitration.

Prostacyclin (PGI(2)) synthase is a heme-thiolate (P450) protein which reacts with low levels of peroxynitrite (PN) under tyrosine nitration and inactivation. Studying heme proteins as models, we have found the heme-thiolate protein NADH-NO reductase (P450(NOR)) to be highly efficient in decomposing PN under concomitant nitration of phenol. The present study investigates two other P450 proteins, P450(BM-3) and chloroperoxidase, in order to test for the specific role of the thiolate ligand in the reaction with PN. A comparison with horseradish peroxidase and microperoxidase gives evidence of kinetic differences that classify heme-thiolate proteins, but not other heme proteins, as effective inhibitors of PGI(2) synthase nitration and inactivation. P450(BM-3) with PN catalyzes phenol nitration and nitration of its own tyrosine below 10 microM PN, whereas chloroperoxidase and P450(NOR) at such concentrations also nitrate phenol but not enzyme-bound tyrosine residues. We conclude that heme-thiolate proteins in general exhibit high reactivity with PN and turnover, probably due to the special electronic structure of the presumed thiolate-ferryl intermediate.

Proton delivery in NO reduction by fungal nitric-oxide reductase. Cryogenic crystallography, spectroscopy, and kinetics of ferric-NO complexes of wild-type and mutant enzymes.

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the reduction of NO to N(2)O through its ferric-NO complex, the first intermediate of the catalysis. Crystal structures of the ferric-NO forms of wild type (WT) fungal NOR, and of the Ser(286) --> Val and Ser(286) --> Thr mutant enzymes were determined to 1.7-A resolution at cryogenic temperature (100 K). This shows a slightly tilted and bent NO binding to the heme iron, in sharp contrast to the highly bent NO coordination found in ferrous hemoproteins. In the WT structure, a specific hydrogen-bonding network that connects the active site to the solvent was identified, H(2)O(Wat(74))-Ser(286)-H(2)O(Wat(33))-Asp(393)-solvent. Wat(74) is located 3.10 A from the iron-bound NO. Replacement of Ser(286) with Val or Thr scarcely alters the NO coordination structure but expels the water molecules, Wat(74) from the active site. The Asp(393) mutation does not influence the position of Wat(74), but disrupts the hydrogen-bonding network at Wat(33), as evidenced by enzymatic, kinetic, and spectroscopic (resonance Raman and IR) results. The structural changes observed upon the Ser(286) or the Asp(393) mutation are consistent with the dramatic loss of the enzymatic activity for the NO reduction of fungal NOR. We have conclusively identified the water molecule, Wat(74), adjacent to the iron-bound NO as a proton donor to the Fe-NO moiety. In addition, we find the hydrogen-bonding network, H(2)O(Wat(74))-Ser(286)-H(2)O(Wat(33))-Asp(393), as a proton delivery pathway in the NO reduction reaction by fungal NOR.

Mutation effects of a conserved threonine (Thr243) of cytochrome P450nor on its structure and function.

Threonine 243 of cytochrome P450nor (fungal nitric oxide reductase) corresponds to the 'conserved' Thr in the long I helix of monooxygenase cytochrome P450s. In P450nor, the replacement of Thr243 with Asn, Ala or Val makes the enzymatic activity dramatically reduce. In order to understand the roles of Thr243 in the reduction reaction of NO by P450nor, the crystal structures of three Thr243 mutants (Thr243-->Asn, Thr243-->Val, Thr243-->Ala) of P450nor were determined at a 1.4-A resolution and at cryogenic temperature. However, the hydrogen-bonding pattern in the heme pocket of these mutants is essentially similar for that of the WT enzyme. This suggests that the determination of the structure of the NADH complex of P450nor is required, in order to evaluate the role of Thr243 in its enzymatic reaction. We attempted to crystallize the NADH complex under several conditions, but have not yet been successful.

Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

Peroxynitrite reaction with heme proteins.

We have reported that low levels of peroxynitrite (PN) can cause inactivation of the heme-thiolate protein prostacyclin (PGI2)-synthase by nitration of a tyrosine residue. To prove that iron catalysis is involved we studied the interaction of PN with microperoxidase and P450nor, a heme-thiolate protein of known structure. Spectral and kinetic analyses allow to conclude on a ferryl nitrogen dioxide complex as an intermediate which decomposes in the presence of an excess of PN under formation of dioxygen, nitrite, and nitrate. This occurs in a catalytic cycle which was more efficient with P450nor than with microperoxidase. If phenol was added to the reaction mixtures of PN and the ferric complexes the ratio of hydroxylated to nitrated phenols decreased compared to the metal-free system. Phenol competed with the formation of dioxygen indicating that the ferryl intermediate was involved in both pathways. One therefore can postulate that the ferryl complex reacts with phenol to give the phenoxyradical which is nitrated in the presence of nitrogen dioxide but does not give hydroxylated products as with metal-free PN. Alternately, the ferryl nitrogen dioxide complex can oxidize a second PN molecule to the radical, *OONO, which can decompose to dioxygen and NO. The latter forms N2O3, with the remaining *NO2 radical. A third pathway consists in the isomerization to nitrate which also is catalyzed by the heme proteins since the ratio of nitrite/nitrate does not change significantly during the catalytic reaction with excess of PN. Our data explain the mechanism of nitration of PGI2-synthase, suggest a role of P450nor as a PN scavenger, and favor heme-thiolate complexes for trapping PN.

Denitrification by yeasts and occurrence of cytochrome P450nor in Trichosporon cutaneum.

Yeasts of various genera were screened for denitrifying activity, and several yeast strains such as Trichosporon cutaneum, Fellomyces fuzhouensis, and Candida sp. were found to exhibit distinct activities to convert nitrite to nitrous oxide. Dissimilatory nitrite reductase (Nir) or nitric oxide reductase (Nor) activities were detected in the cell-free extracts of these yeasts. Spectrophotometric as well as Western blot analyses showed that T. cutaneum contains Nor of the cytochrome P450 type. This is the first report that shows that denitrification is also distributed among yeasts although their systems are incomplete, only capable of reducing nitrite to nitrous oxide.

Denitrification by actinomycetes and purification of dissimilatory nitrite reductase and azurin from Streptomyces thioluteus.

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N2O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N2O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.

Site-directed mutagenesis of the conserved threonine (Thr243) of the distal helix of fungal cytochrome P450nor.

Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduction in denitrifying fungi. Threonine 243 (Thr243) of P450nor, which corresponds to the conserved threonine of monooxygenase cytochrome P450s, was replaced by 18 different amino acids via site-directed mutagenesis. The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligation, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural changes in the heme environment on Thr243 mutation. However, the NO reduction activity was dramatically altered by Thr243 mutation, depending on the properties of the replaced amino acids. The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retained on substitution of Asn, Ser, and Gly for Thr243, while it was profoundly decreased or lost on substitution with other amino acids. Kinetic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainly results from a decrease in the rate of reduction of the ferric-NO complex with NADH. On the basis of these enzymatic, kinetic, and spectroscopic results, as well as on the basis of the crystal data for native P450nor [Park, S.-Y., et al. (1997) Nat. Struct. Biol. 4, 827-832], the role of the conserved threonine at the 243 position in the NO reduction reaction by P450nor is discussed. We also discuss structural similarities or differences in the vicinity of the conserved threonine between P450nor and other monooxygenase P450s.