Biomedical subjects

H Shoun

Publications and source records attributed to H Shoun.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Catalysis↗

PubMed1998

The effects of heme modification on reactivity, ligand binding properties and iron-coordination structures of cytochrome P450nor.

Artificial cytochrome P450nors (nitric oxide reductase) were prepared by replacing the native protoheme with various 2,4-substituted hemes: meso-, deutero-, and diacetyldeutero-hemes. For these samples, the ratio of low spin/high spin states of the ferric resting enzyme were varied, indicating that the coordination of the water molecule at the iron sixth site was affected by the electron withdrawing capacities of the heme 2,4-substituents. The binding of the water molecule reduces the rate of binding of nitric oxide (NO) to the ferric iron. In addition, the reduction reaction of the ferric-NO complex with NADH, which constitutes the second step in the NO reduction, was facilitated by the electron withdrawing capacity of 2,4-substituents. Consequently, proto- (native-) P450nor exhibited the highest overall enzymatic activity (NO reduction activity), while the enzymes containing diacetyl-, deutero-, and meso-hemes had considerably lower activities, since the NO reduction activity is determined by a balance of the reaction rates of the above two steps. The optical absorption spectra of the ferric-NO and the ferrous-CO complexes of the reconstituted enzymes show that the electron density on the heme in both states was modulated by the substituent groups. However, the resonance Raman spectral measurements showed that the Fe-NO and N-O stretching frequencies in the ferric-NO complex were insensitive to the electron density of the heme while the Fe-CO and C-O stretching frequencies in the ferrous-CO complex were sensitively varied by the electron withdrawing capacity of the 2,4-substituent. The differences are discussed in terms of the difference in the iron-ligand bond characters between the ferric-NO and the ferrous-CO complexes.

Binding Sites↗

PubMed1998

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.

Ascomycota↗

PubMed1997

Purification and characterization of a flavohemoglobin from the denitrifying fungus Fusarium oxysporum.

A flavohemoprotein was purified to homogeneity from the denitrifying fungus Fusarium oxysporum. The purified protein existed as a monomer with a molecular weight of 44 kDa. It was purified in an oxidized form and exhibited the absorption maxima at 401, 540 and 643 nm in its resting form, and at 434 and 555 nm upon reduction with dithionite, respectively. The protein contained 0.5 mol protoheme/mol and 1.1 mol FAD/mol, respectively. When the resting flavohemoprotein was aerobically incubated with NAD(P)H, it was converted to a spectral species that is spectrally very similar to oxyhemoglobins. These properties are characteristics of flavohemoglobins (FHb) of Alcaligenes eutrophus, Escherichia coli, and baker's yeast. Further the amino terminal amino acid sequence of the protein of F. oxysporum was similar to those of these FHbs. These results suggest that the isolated flavohemoprotein of F. oxysporum would be a counterpart of the proteins in the FHb family.

Amino Acid Sequence↗

PubMed1997

Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor.

Cytochrome P450nor (P450nor) is a heme-containing nitric oxide reductase from the denitrifying fungus, Fusarium oxysporum. This enzyme catalyzes the reduction of NO to N2O. In the present study, we report results from preliminary crystallographic and electron paramagnetic resonance (EPR) analysis of a single crystal of P450nor. The crystal was grown in 100 mM MES buffer at pH 5.6 using PEG 4000 as a precipitant. It belongs to the orthorhombic system with cell dimensions of a = 54.99 A, b = 82.66 A, c = 87.21 A, and the space group is P2(1)2(1)2(1). The crystal diffracts synchrotron radiation at higher than 2.0 A resolution, and therefore it is suitable for X-ray crystal structure analysis at atomic resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the heme iron. The structure solution is currently underway by means of MIR and MAD techniques. EPR analysis determined the orientation of the heme within the P450nor crystal.

Crystallography, X-Ray↗

PubMed1997

Functional and structural comparison of nitric oxide reductases from denitrifying fungi Cylindrocarpon tonkinense and Fusarium oxysporum.

Two isozymes of nitric oxide reductase (Nor) from the denitrifying fungus Cylindrocarpon tonkinense (c.Nor1 and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883-889). However, they catalyze the NO reduction to N2O, but not the monooxygenation reaction using O2. We kinetically and spectrophotometrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapid scan methods. The enzyme in the Fe3+ state can bind NO to yield the Fe3+ NO complex. When the resultant Fe3+ NO complex reacted with the electron donor, it was converted to the Fe3+ enzyme via a transient formation of the characteristic intermediate (I). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shiro et al. (1995) J. Biol. Chem. 270, 1617-1623), suggesting that these fungal Nor's catalyze the NO reduction by the same mechanism. Most probably, the Fe3+ NO complex of the Nor is reduced with two-electrons directly transferred from NAD(P)H to yield the intermediate I, and then the I reacts with another NO to generate N2O and the Fe3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Nor1 + NADH, c.Nor2 + NADPH, c.Nor2 + NADH and f.Nor + NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe3+ NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted 1H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state, where the iron-bound Cys beta-CH2 signal was observed at the same position (approximately 270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S-) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopic data, we suggested that the Fe-S- binding character is not essential for the NO reduction reactivity, but that the subtle difference in interaction of their hemes with the surroundings is possibly responsible for the difference in the Nor reactivity, especially in the electron transfer step from NAD(P)H to the Fe3+ NO moiety.

Basidiomycota↗

PubMed1997

Fungal P450nor: expression in Escherichia coli and site-directed mutageneses at the putative distal region.

P450nor, nitric oxide reductase from Fusarium oxysporum, was expressed in the soluble fraction of Escherichia coli cells by modifying the N-terminal codons and utilizing the pCW vector. The modified P450nor purified to electrophoretic homogeneity had spectral and enzymatic properties identical to those of native P450nor obtained from the fungi. Residues 239 to 247 of the modified P450nor were replaced with Lys by site-directed mutagenesis. Thr-243 to His- and Arg-mutants were also created. Among 11 mutants, only the Thr-243 to Lys-mutant exhibited an absorption spectrum characteristic of a nitrogenous ligand-bound form of P450 at pH 8.0 in the ferric state, but the spectrum was altered to that of the wild-type P450nor as the pH was lowered. Other mutants had spectra typical of the low- and high-spin mixed form of P450 in the ferric state. In the ferrous state, all mutants showed the same spectrum as the wild-type P450nor. Nitric oxide reductase activity was considerably decreased by the replacement of Thr-243 with Lys, His, or Arg or Ala-239 with Lys. These findings indicate that Thr-243 is located more closely to the heme iron than other residues in the putative distal helix of P450nor and plays an important role in the catalytic activity, but a specific difference in the structure of the heme pocket from other P450s is suggested.

Amino Acid Sequence↗

PubMed1997

Absorption spectral studies on heme ligand interactions of P-450nor.

Heme-external ligand interactions of P-450nor were examined spectrophotometrically and compared with those of other P-450s. Most nitrogenous ligands induced type II spectral changes on binding to ferric P-450nor, as did other P-450s. In contrast with other P-450s, 2-methylpyridine and 1-butanol induced type I changes in the spectrum of P-450nor. No spectral interaction of ferrous P-450nor with these ligands was observed. The absorption spectra of the alkyl isocyanide complexes of ferrous P-450nor exhibited the Soret peak at 427 nm with a slight shoulder at around 455 nm at neutral pH, and this shoulder was intensified as the pH was increased, suggesting that the isocyanide complexes of P-450nor existed in two states (the 430 and 455 nm states) which were in pH-dependent equilibrium in a similar manner to microsomal P-450s. However, the equilibrium was shifted mostly to the 430 nm state in the complexes of P-450nor. The findings suggest that P-450nor, especially its ferrous form, has some distinct features from P-450cam and microsomal P-450s in the distal heme environment.

Binding, Competitive↗

PubMed1997

Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

Amino Acid Sequence↗

PubMed1996

Denitrification, a novel type of respiratory metabolism in fungal mitochondrion.

Subcellular localization and coupling to ATP synthesis were investigated with respect to the denitrifying systems of two fungi, Fusarium oxysporum and Cylindrocarpon tonkinense. Dissimilatory nitrate reductase of F. oxysporum or nitrite reductase of C. tonkinense could be detected in the mitochondrial fraction prepared from denitrifying cells of each fungus. Fluorescence immunolocalization, cofractionation with mitochondrial marker enzymes, and cytochromes provided evidence that the denitrifying enzymes are co-purified with mitochondria. Respiratory substrates such as malate plus pyruvate, succinate, and formate were effective donors of electrons to these activities in the mitochondrial fractions. Moreover, nitrite and nitrate reduction were shown to be coupled to the synthesis of ATP with energy yields (P:NO3- or P:2e ratios) of 0.88 to 1.4, depending upon whether malate/pyruvate or succinate were provided as substrates. Nitrate or nitrite reductase activity was inhibited by inhibitors such as rotenone, antimycin A, and thenoyltrifluoroacetone. Thus, fungal denitrification activities are localized to mitochondria and are coupled to the synthesis of ATP. The existence of these novel respiration systems are discussed with regard to the origin and evolution of mitochondria.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Two isozymes of P450nor of Cylindrocarpon tonkinense: molecular cloning of the cDNAs and genes, expressions in the yeast, and the putative NAD(P)H-binding site.

The cDNAs and genes for two isozymes of cytochrome P450nor of the fungus Cylindrocarpon tonkinense, P450nor1 and P450nor2, were cloned and sequenced. Their deduced amino acid sequences respectively showed 83 and 70% identity to that of P450nor of Fusarium oxysporum, and 69% identity to each other. The genes for P450nor1 and P450nor2 were termed, respectively, CYP 55A2 and CYP 55A3. The cDNA for P450nor1 contained a targeting-like presequence upstream the N-terminus of mature protein whereas that for P450nor2 did not, suggesting their different intracellular localisations. We also succeeded in expressing these P450nor isoforms in the host-vector system of the yeast Saccharomyces cerevisiae. We purified one of the recombinant proteins, P450nor of F oxysporum. Little difference could be observed between the native and recombinant proteins in catalytic and spectroscopic properties. We constructed chimeric proteins of P450nor of F oxysporum and P450nor2 which are different in their specificity against the electron donors: reduced pyridine nucleotides. The specificity of chimeric proteins against NADH/NADPH showed that the specificity is determined by the N-terminal half of protein. We found a consensus amino acid sequence between three isoforms of P450nor, A-X-G-X-X-A, similar to the NAD-binding motif G-X-G-X-X-G/A in the region that corresponds to the B'-helix in P450cam.

Amino Acid Sequence↗

PubMed1996

Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum.

We have purified membrane-bound fatty acid (omega-1-omega-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent M(r) value of 118,000. The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase. These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase. Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical. Further, the fungal hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart. On the other hand, a unique phenomenon was found with the fungal hydroxylase: its NADPH-cytochrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid). This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450. These results raise interesting questions about the molecular evolution of P450. Here we term the fungal hydroxylase cytochrome P450foxy.

Blotting, Western↗

PubMed1996

N-terminal processing and amino acid sequence of two isoforms of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum.

Cytochrome P450nor (P450nor), a nitric oxide reductase involved in the denitrifying system of the fungus Fusarium oxysporum, revealed molecular multiplicity. Two isoforms of P450nor, termed P450norA (norA) and P450norB (norB), were isolated. They had distinct isoelectric points of 5.1 (norA) and 4.9 (norB). However, their catalytic, spectroscopic, and immunological properties were almost identical. Partial amino acid sequences, involving 263 amino acid residues of norA and 278 residues of norB among 404 and 402 residues, respectively, were determined. Corresponding sequences in the isoforms were identical, and all of the determined partial sequences of norA or norB coincided with the sequence deduced from the CYP 55 gene or its cDNA. The amino acid sequence determination ruled out the possibility that there is a redox center in P450nor derived from amino acid residues, e.g., quinonoid cofactors. The only difference between norA and norB was in their N-termini. The N-terminus of norA was a threonine residue, whereas that of norB was an N-acetylated alanyl residue and norB was shorter by 2 residues than norA. The results suggested that norA and norB may be the products of the same gene, but translated from different initiation codons. The hypothetical precursor of norA would have a presequence containing targeting and sorting signals for transportation to the intermembrane space of mitochondria. This is consistent with the results of a Western-blot analysis which showed that norA was recovered only in particulate fractions, whereas norB was in the soluble fraction. It is therefore likely that the intracellular localizations as well as the N-termini of norA and norB are different, owing to the differences in the translational initiation codons and co/post-translational processings.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of an intracellular alpha-D-xylosidase from Penicillium wortmannii IFO 7237.

Intracellular alpha-D-xylosidase from Penicillium wortmannii IFO 7237 was obtained by grinding the mold with almina in phosphate buffer, and the cell-free extract was purified to a homogeneous state on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was estimated to be 290,000 by gel filtration chromatography (Superdex 200) and 73,000 was obtained by SDS-PAGE. The purified alpha-D-xylosidase had an isoelectric point at pH 5.0. The optimum activity for the enzyme was found to be at pH 6.5 and 45 degrees C. The enzyme showed a hydrolytic activity on p-NO2-phenyl-alpha-D-xylopyranoside (alpha-p-NPX) while methyl-alpha-D-xylopyranoside (alpha-MX) was not hydrolyzed at all. It also showed lower activity for xyloglucan oligosaccharides. The apparent Km values of the enzyme for alpha-p-NPX and isoprimeverose were 1.9 mM and 50 mM, respectively.

Glucans↗

PubMed1995

Iron-ligand structure and iron redox property of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum: relevance to its NO reduction activity.

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements. The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S(-)-Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not. In spite of the same S(-)-Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum.

A copper-containing nitrite reductase (Cu-NiR) was purified to homogeneity from the denitrifying fungus Fusarium oxysporum. The enzyme seemed to consist of two subunits with almost the same M(r) value of 41,800 and contains two atoms of copper per subunit. The electron paramagnetic resonance spectrum showed that both type 1 and type 2 copper centers are present in the protein, whereas the visible absorption spectrum exhibited a sole and strong absorption maximum at 595 nm, causing a blue but not green color. The reaction product due to the Cu-NiR was mainly nitric oxide (NO), whereas a stoichiometric amount of nitrous oxide (N2O) was formed when cytochrome P-450nor was further added to the assay system. Therefore, the denitrifying (N2O forming) nitrite reductase activity that we had detected in the cell-free extract of the denitrifying cells (Shoun, H., and Tanimoto, T. (1991) J. Biol. Chem. 266, 11078-11082) could be reconstituted upon combination of the purified Cu-NiR and P-450nor. The Km for nitrite and specific activity at pH 7.0 were estimated as 49 microM and 447 mumol NO.min-1.mg protein-1, respectively. Its activity was strongly inhibited by cyanide, carbon monoxide, and diethyldithiocarbamate, whereas enormously restored by the addition of cupric ions. An azurin-like blue copper protein (M(r) = 15,000) and a cytochrome c were also isolated from the same fungus, both of which together with cytochrome c of the yeast Saccharomyces cerevisiae were effective in donating electrons to the fungal Cu-NiR. The result suggested that the physiological electron donor of the Cu-NiR is the respiratory electron transport system. The intracellular localization of Cu-NiR was investigated, and it was suggested that the Cu-NiR localizes in an organelle such as mitochondrion. These findings showed the identity in many aspects between the fungal nitrite reductase and bacterial dissimilatory Cu-NiRs. This is the first isolation of dissimilatory NiR from a eukaryote.

Bacterial Proteins↗

PubMed1995

Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism.

Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+--> N2O + H2O + NAD+). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = approximately 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)2-complex. We determined the rate constants of these reactions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M-1 s-1), the NADH reduction of P450nor(Fe3+NO) (0.9 x 10(6) M-1 s-1), and the spontaneous decomposition of the intermediate (0.027 s-1). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s-1 at 10 degrees C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.

Cytochrome P-450 Enzyme System↗

PubMed1995

Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor.

We examined the denitrification system of the fungus Cylindrocapon tonkinense and found several properties distinct from those of the denitrification system of Fusarium oxysporum. C. tonkinense could form N2O from nitrite under restricted aeration but could not reduce nitrate by dissimilatory metabolism. Nitrite-dependent N2O formation and/or cell growth during the anaerobic culture was not affected by further addition of ammonium ions but was suppressed by respiration inhibitors such as rotenone or antimycin, suggesting that denitrification plays a physiological role in respiration. Dissimilatory nitrite reductase and nitric oxide reductase (Nor) activities could not be detected in cell extracts of the denitrifying cells. The Nor activity was purified and found to depend upon two isoenzymes of Cytochrome P-450nor (P-450nor), which were designated P-450nor1 and P-450nor2. These isozymes differed in the N-terminal amino acid sequence, isoelectric point, specificity to the reduced pyridine nucleotide (NADH or NADPH), and the reactivity to the antibody to P-450nor of F. oxysporum. the difference between the specificities to NADH and NADPH suggests that P-450nor1 and P-450nor2 play different roles in anaerobic energy acquisition.

Amino Acid Sequence↗