Catalatic Activity Research Articles

Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H

Catalases are essential components of the cellular equipment to cope with oxidative stress. We have purified and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H. This oxidative stress-induced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid. We have used consecutive steps of hydrophobic, molecular sieve, and ion exchange chromatography to achieve a high state of purity for this metalloenzyme. The purified sample of catalase exhibited a specific catalatic activity of 55,900 U/mg, allosteric behavior in peroxidic reaction, a broad pH optimum, and a rather atypical electronic spectrum. The sample of highest purity was subjected to mass spectrometry analysis. The molecular weight of the subunit of this homodimeric protein was determined as 55,417 Da. The Qq-TOF mass analysis method allowed us to sequence short tryptic fragments of this catalase. Five such fragments with a total length of 57 amino acids together with several enzymatic properties allowed the classification of this hydroperoxidase as belonging to clade III of monofunctional catalases. The highest sequence similarity is with the catalase from Vibrio fischeri. The presented results imply the significance of this inducible enzyme in the prevention of toxic effects of oxidative stress for bacterial cells.

Protein-based radicals in the catalase-peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site.

Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.

UVB Light Stimulates Production of Reactive Oxygen Species: UNEXPECTED ROLE FOR CATALASE

In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320-360 nm range with additional soret peaks around 400-410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer.

Relative contributions of heart mitochondria glutathione peroxidase and catalase to H(2)O(2) detoxification in in vivo conditions.

This study was aimed at assessing the relative contributions to H(2)O(2) detoxification by glutathione peroxidase and catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order to minimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities were determined along with a simulation profile. Results show that the contribution of catalase to H(2)O(2) removal in heart mitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusion and involving extensive glutathione depletion and high H(2)O(2) concentrations. Conversely, maintenance of the steady state levels of H(2)O(2) in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggested that the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic rather than catalatic activity.

Modulation of catalase peroxidatic and catalatic activity by nitric oxide

Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch. Biochem. Biophys. 316:327–334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide. Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide.

Fully-automated spectrophotometric method for measurement of antioxidant activity of catalase

Objectives: Develop fully automated assay of antioxidant catalatic activity of catalase. Design and methods: The assay is based on standard, clinical chemistry automated analyzer methods for measuring hydrogen peroxide by using the Trinder reagent. Catalase competes with 324 U/L horseradish peroxidase (type XII) and Trinder reagent for hydrogen peroxide produced by 46 U/L uricase action on urate. Unit activity is defined as 50% inhibition of maximal color development. Results: Within-run coefficients of variation (cv) were 2% for standards and samples, whereas between-run cv was 3.1% for standards and 7.3% for samples. Dilutional parallelism and linearity were demonstrated for 8-fold dilutions of samples over the range 0.1 to 1.1 U/mL. Recovery of added catalase was complete. Samples are stable to freezing and storage for 1 week at −80 °C. Activities (units/mL) ranged from 0.29 to 0.41 in human and canine plasma, and for erythrocytes from 48 to 70 in man, 17 to 19 in dogs, and 60 to 89 in rats. Rat liver activity (units/g wet weight) was age-dependent and ranged from 17 to 24 at 2 months, and from 19 to 37 at 6 months. Conclusion: The first, fully automated assay for the measurement of catalatic activity of catalase in plasma, erythrocytes, and liver is demonstrated for multiple species. The assay is simple, precise, relatively inexpensive, and rapid.

A Possible Role of the Na+/K+-ATPase for O2 Production from H2O2

We are attempting to supply a new insight on interaction between Na+/K+-ATPase and H2O2. We demonstrate that in vitro the Na+/K+-ATPase, a non heme-protein, is able to disproportionate H2O2 catalatically into dioxygen and water, as well as C40 catalase. By polarography, we quantify O2 production and by Raman spectroscopy H2O2 consumption. A comparative analysis of kinetics parameters relative to O2 production shows that for Na+/K+-ATPase the affinity of the catalytic site able to transform H2O2 into O2 is twice weaker than that for C40 catalase. It also shows that the molar activity for O2 production is 300-fold weaker for ATPase than for catalase. Inhibitors, pH and GSH studies highlight the differences between the heme- and nonheme-proteins. Indeed, for C40, NaN3 is strongly inhibiting, but much less for ATPase. The pH range for the catalatic activity of ATPase is wide (6.5 to 8.5), while it is not for C40 catalase (optimum at pH 8). The Na+/K+-ATPase catalatic activity is reduced in presence of glutathione, while it is not the case with C40 catalase.

Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis.

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I.

Copper–Adenine Catalyst for O2 Production from H2O2

In solutions of CuCl2 and adenine copper can be bound to adenine. Two Cu(adenine)2 complexes [Cu(C5H5N5)2]2+/Cu(C5H4N5)2] are in equilibrium with free adenine. Copper–adenine complexes present a catalatic activity (e.g., H2O2 disproportionation into O2 and water) but depending on complex concentration H2O2 also strongly oxidizes the adenine within the complexes. Raman spectroscopy quantifies copper–adenine complex formation and H2O2 consumption; polarography quantifies O2 production. As for C40 catalase, optimal catalatic capacities depend on physiological conditions, such as pH and temperature. The comparative analysis of kinetic parameters shows that the affinity for H2O2 of Cu(adenine)2 is 37-fold lower than that of C40 catalase and that the molar activity for O2 production is 200-fold weaker for Cu(adenine)2 than for the enzyme. In the 10−6–10−3 M range, the strong decrease of activity with raising complex concentration is explained by aggregation or stacking, which protects Cu(adenine)2 entities from H2O2 oxidation, but also decreases O2 production.

Structure of catalase-A from Saccharomyces cerevisiae

The structure of the peroxisomal catalase A from the budding yeast Saccharomyces cerevisiae, with 515 residues per subunit, has been determined and refined to 2.4 Å resolution. The crystallographic agreement factors R and R free are 15.4 % and 19.8 %, respectively. A tetramer with accurate 222-molecular symmetry is located in the asymmetric unit of the crystal. The conformation of the central core of catalase A, about 300 residues, remains similar to the structure of catalases from distantly related organisms. In contrast, catalase A lacks a carboxy-terminal domain equivalent to that found in catalase from Penicillium vitalae, the only other fungal catalase structure available. Structural peculiarities related with the heme and NADP(H) binding pockets can be correlated with biochemical characteristics of the catalase A enzyme. The network of molecular cavities and channels, filled with solvent molecules, supports the existence of one major substrate entry and at least two possible alternative pathways to the heme active site. The structure of the variant protein Val111Ala, also determined by X-ray crystallography at 2.8 Å resolution, shows a few, well-localized, differences with respect to the wild-type enzyme. These differences, that include the widening of the entry channel in its narrowest point, provide an explanation for both the increased peroxidatic activity and the reduced catalatic activity of this mutant.

Scavenging of superoxide and hydrogen peroxide in blue‐green algae (cyanobacteria)

Blue‐green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant‐type photosynthetic organisms. Within a single prokaryotic cell, they have uniquely accommodated both oxygenic photosynthesis and aerobic respiration, which are known to produce superoxide and hydrogen peroxide as inevitable byproducts. Two types of superoxide dismutase have been characterized in both N2‐fixing and non‐N2‐fixing cyanobacteria, namely cytosolic iron‐containing superoxide dismutase and thylakoid‐bound manganese‐containing superoxide dismutase. No qualitative differences between various cell types (vegetative cells, heterocysts) were found. In contrast to chloroplasts, most of the cyanobacterial species show catalatic activity. From two species the corresponding enzymes have been characterized as typical prokaryotic (bifunctional) catalase‐peroxidases with homologies to cytochrome c peroxidases and ascorbate peroxidases. In addition to catalatic activity, some strains exhibit ascorbate peroxidase activity, but to date there are no reports detailing purification and characterization.Cyanobacteria were found to contain low intracellular ascorbate concentrations (30‐100 µM) and 2‐5 mM glutathione. Both monodehydroascorbate and glutathione reductase activities were detected in most species examined, whereas dehydroascorbate reductase activity was absent. The question as to whether a glutathione‐ascorbate cycle exists in cyanobacteria cannot be answered at present.

Mechanisms for Protection against Inactivation of Manganese Peroxidase by Hydrogen Peroxide

It has been reported that cation radicals of aromatic substrates maintain the active form of lignin peroxidase by oxidatively converting compound III, generated during peroxidase turnover, into ferric enzyme (D. P. Barr and S. D. Aust, 1994,Arch. Biochem. Biophys.312, 511–515). In this work, we investigated protective mechanisms for manganese peroxidase. Oxidation of Mn(II) by manganese peroxidase displayed complex kinetics, which were explained by accumulation of compound III followed by its reactivation by the enzymatically produced Mn(III). Conversion of compound III to ferric enzyme by Mn(III) was not observed for lignin peroxidase or heme propionate-modified recombinant manganese peroxidase, suggesting that Mn(III) may interact with compound III of native manganese peroxidase at a heme propionate to oxidize iron-coordinated superoxide via long-range electron transfer. Additionally, Mn(II) also reactivated compound III. Although this reaction was slower, it could prevent compound III accumulation when excess Mn(II) was present. Another protective mechanism for manganese peroxidase is proposed for insufficient chelator conditions. In contrast to effective Mn(II) chelators, low-affinity ligands supported considerably slower enzyme turnover, and Mn(III) released was more reactive with hydrogen peroxide, resulting in a catalase-type reaction. Reactivation of compound III and catalatic activity may provide biologically relevant mechanisms for protection of manganese peroxidase against suicidal inactivation by hydrogen peroxide under a variety of manganese and oxalate conditions.

Catalase-peroxidase of Caulobacter crescentus: function and role in stationary-phase survival.

Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment. Physiological studies of oxidative and starvation stresses in C. crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase. The katG gene was shown to be solely responsible for catalase and peroxidase activity in C. crescentus. Like the katG of Escherichia coli, C. crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate. However, dramatic differences are seen in growth stage induction. E. coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase. In contrast, C. crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase. Moreover, the survival of a C. crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E. coli katE and katG null mutants. These results indicate a major role for C. crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.

Effects of Mn2+and Oxalate on the Catalatic Activity of Manganese Peroxidase

Manganese peroxidase fromPhanerochaete chrysosporiumis an extracellular heme-containing enzyme known to catalyze the oxidation of Mn2+to Mn3+in a reaction requiring oxalate or another appropriate manganese chelator. We have found that the enzyme can also catalyze a manganese-dependent disproportionation of hydrogen peroxide when a manganese chelator is not included. The catalatic activity was observed in the pH range from 3.0 to 8.5, and the apparent second-order rate constant for catalatic reaction was about 2 × 105M−1s−1at pH 4.5 to 7.0 at 25°C. Oxalate inhibited oxygen production by increasing the apparent Kmfor Mn2+for catalatic activity from micromolar to millimolar levels and facilitating peroxidase activity. Catalase-type function was recovered by excess of Mn2+in the presence of oxalate. We propose that catalatic activity may protect the enzyme from inactivation by hydrogen peroxide in an environment where free oxalate may be limited.

Nucleotide Pools and Physiological Consequences of Heat Shock in Euglena Cells

Etiolated cells of Euglena gracilis , grown at 26°C in the dark, were taken at different growth phases and submitted to heat shocks (up to 2 hr) at temperatures ranging from 33 to 42°C. Their volumes and mortality increased rapidly with temperature and the duration of the shock. On the contrary, the apparent gas exchange during respiration slowed suddenly at temperatures higher than 38°C. A direct and positive correlation appears to exist between high Energy Charges, catalatic activities, and the persistence of respiration. After only 1 hr at 40 or 42°C, respiration ceased, while cell catalatic activities and Energy Charge collapsed. The adenylic pool of treated cells increased independently of the growth phase of cells. This evolution was parallel to that of cell volume. These results are discussed with respect to variations in catalatic activity.

Fat and Water Composition Affects Residual Catalatic Activity in Chicken Breast Heated to Specific End‐point Temperatures

ABSTRACTFat and water contents of meat tissues strongly influence their thermo‐physical properties. Seven groups of ground chicken breast meat containing varying amounts of fat and water were prepared for this investigation. Each group was heated to end‐point temperatures (EPTs) of 69.0 to 71.0°C at 0.5°C intervals. Severity of heat treatments was evaluated by the total process lethality calculated using the General Method. The fat and water contents affected the heating time to reach specific EPTs and the residual catalatic activity at EPTs of 69.0, 69.5 and 70.0°C, but did not affect total process lethality. The variation of the catalase method for determining EPT was related to the sample composition.

CATALATIC ACTIVITY AS AN INDICATOR OF END‐POINT TEMPERATURES IN BREADED, HEAT PROCESSED CHICKEN PATTIES

ABSTRACTAdequate heat‐processing of meat and poultry products to destroy harmful pathogens is important to the consumer's safety. The catalatic activity, defined as the decomposition of H2O2 into H2O and O2, has been evaluated as a potential indicator of heat treatment adequacy for chicken patties. Commercially prepared breast and leg meat patties were processed through a belt type grill to various end‐point temperatures (EPTs) and their catalatic activities monitored for up to 120 min. All breast samples heated to EPTs of ≤ 69C and all leg samples heated to ≥ 68C showed positive reactions after incubation for 10 min. As the EPT increased, fewer samples were positive, indicating a decrease in the catalatic activity. For EPTs of ≥ 73C, three of 12 breasts and one of 17 leg patties showed positive activity after 30 min incubation. The catalatic activity test may offer a simple and rapid procedure for estimating the EPT at 73C with 3‐4C variation for commercially processed chicken patties.

Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals.

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H2O2, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H2O2 resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H2O2 showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H2O2. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalytic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10(4) fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: approximately 20 mol H2O2 and abolished the protein radical steady state. The biological implications for these results are discussed.

Comparison of Isoniazid Oxidation Catalyzed by Bacterial Catalase–Peroxidases and Horseradish Peroxidase

The physical properties and activities of the purified catalase–peroxidase hydroperoxidase I (HPI) ofEscherichia coli(EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxidase (HRP). The catalase–peroxidase proteins had similar absorption spectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e resulted in the formation of an active catalase–peroxidase lacking the carboxyl-terminal extension. The three enzymes, HRP, MtHPI-e, and EcHPI, mediated the isoniazid- and H2O2-dependent production of radical species, as detected by nitroblue tetrazolium reduction. A constant flux of H2O2, generated in situ from glucose oxidase and glucose was used. MtHPI-e was more effective at isoniazid-dependent radical production than EcHPI and HRP. Similar qualitative results were obtained by staining nondenaturing polyacrylamide gels for activity with nitroblue tetrazolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2consistent with the formation of several typical reaction intermediates, whereas the catalase–peroxidases exhibited no distinct spectral changes. The results suggest that the sensitivity of M. tuberculosis to isoniazid may be the result of isoniazid-dependent radical formation by the catalase–peroxidase in the absence of other catalase activities to remove substrate H2O2.

Crystal Structure ofProteus mirabilisPR Catalase With and Without Bound NADPH

A catalase from a peroxide resistant mutant ofProteus mirabilisbinds NADPH tightly. Interestingly, this enzyme can be stripped of NADPH without loss of the catalatic activity. It is the only known non-mammalian catalase able to bind NADPH. The structure without cofactor was solved by molecular replacment using the structure of beef liver catalase as a model. The structure was refined to anR-factor of 19.3% in the range 8 to 2.2 Å resolution. According to the sequence, a methionine sulphone was positioned in the haem active site. The oxidized form of methionine is particular toProteus mirabiliscatalase and likely to produce some steric hindrance in the active site. Two important water molecules are positioned in the haem distal site. These two water molecules are not located in the structure of beef liver catalase, but are supposed to account for the catalytic mechanism. The liganded form was obtained by soaking crystals of the unliganded form into an NADPH solution. The structure was refined to anR-factor of 15.9% in the range of 8 to 3.1 Å resolution using the unliganded structure as a model. The NADPH was clearly located in the electron density map with the same conformation as in beef liver catalase. The NADPH binding induces slight structural changes. However, the imidazole ring of a histidine residue (His284) rotates about 50 ° to accommodate the cofactor. The electron transfer from NADPH to the haem molecule was examined and several pathways are proposed.