Nitric Oxide Reductase Cytochrome P450nor Research Articles

Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.

Hartree–Fock and density functional theory calculations for the reaction mechanism of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum

A reaction mechanism of a nitric oxide reductase, cytochrome P450nor (P450nor) from Fusarium oxysporum, was clarified by using Density functional theory and Hartree–Fock calculations. In this reaction mechanism, molecular orbital (MO) analysis revealed that the NO ligand dissociates from the heme iron immediately after one-electron reduction by NADH, and MO energy analysis revealed that NADH acts as a one-electron reducer, not as a two-electron reducer, and that NADH has a pivotal role different from other one-electron reducers. The role of NADH is to act as a double one-electron donor (i.e. one-electron transfer occurring twice) and to combine with the NO − molecule by charge recombination reaction. Our quantum chemical calculations indicated that all reactions occurring in the heme pocket are too fast to become rate-limiting. Therefore, the rate-limiting steps in the proposed reaction mechanism are the process of capturing NO and NADH into the heme pocket and the process of expelling a product generated in the heme pocket. Kinetics of these processes was discussed based on large-amplitude vibration, which helps capturing and expelling processes in a widely opened heme pocket of P450nor. The reaction mechanism proposed here well explains published experimental data.

3P108 Hartree-Fock and DFT calculations for the reaction mechanism of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum

3P108 Hartree-Fock and DFT calculations for the reaction mechanism of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum

Structural evidence for direct hydride transfer from NADH to cytochrome P450nor.

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover.

X-ray structure of nitric oxide reductase (cytochrome P450nor) at atomic resolution

Crystal structures of the nitric oxide reductase cytochrome P450nor (P450nor) in the ferric resting and the ferrous carbonmonoxy (CO) states have been determined at 1.00 and 1.05 A resolution, respectively. P450nor consists of 403 amino-acid residues (46 kDa) and is one of the largest proteins refined to this resolution so far. The final models have conventional R factors of 10.2% (ferric resting) and 11.7% (ferrous CO), with mean coordinate errors of 0.028 (ferric resting) and 0.030 A (ferrous CO) as calculated from inversion of the full positional least-squares matrix. Owing to the atomic resolution, novel features are found in the refined structures. Firstly, two orientations of the haem are observed both in the ferric resting and the ferrous CO states. Secondly, a disordered water molecule bound to the haem iron is found in the ferric resting state. In addition, the accurate structures at atomic resolution enabled the examination of general stereochemical parameters that are commonly used in refinement cycles of protein structures.

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.

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.

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)

Nitric Oxide Reductase Cytochrome P-450 Gene, CYP 55, of the Fungus Fusarium oxysporum Containing a Potential Binding-Site for FNR, the Transcription Factor Involved in the Regulation of Anaerobic Growth of Escherichia coli1

A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase. The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest. The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced. The P-450nor gene contained 7 introns that consisted of 49 to 55 bp. The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution. Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region. One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria. The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system. Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level. These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite.

Crystallization and preliminary X-ray diffraction studies of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum.

Crystals of cytochrome P450nor (P450nor), nitric oxide reductase from Fusarium oxysporum, were obtained by means of the hanging-drop vapour diffusion technique in the presence of 1.88 M ammonium sulphate (pH 7.2). They belong to the monoclinic space group P2(1), with unit cell dimensions of a = 74.7 A, b = 86.7 A, c = 62.0 A and beta = 97 degrees. There are two molecules of P450nor in the asymmetric unit. The crystal diffracted to beyond 2.5 A resolution and is suitable for X-ray crystallographic study.