REVIEW

Structural domains of P450-containing monooxygenase systems

International Centre for Genetic Engineering and Biotechnology, Area Science Park, 34012 Trieste, Italy and Institute of Biomedical Chemistry, Pogodinskaya 10, 119832 Moscow, Russia

Address (at the moment of publication): Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

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Last modified: Wed Apr 14 11:51:06 BST 2004

Abstract

Introduction

Families of structural domains of P450-containing monooxygenase systems

FAD domain
Ferredoxins
Flavodoxins
Cytochromes b₅
P450 domain

P450 systems in three dimensions

Nitric oxide reductase: homologous but not analogous

Nitric oxide synthase: analogous or homologous?

Intron loss or intron acquisition?

Protein surgery and artificial P450 systems

The P450 Systems on WorldWide Web

Acknowledgements

References

Figures

There exists a widespread division of P450-containing monooxygenase systems into two main types, bacterial/mitochondrial (type I) and microsomal (type II). On the other hand, a principal classification of P450-containing systems was undertaken using the number of their protein components (Degtyarenko and Archakov, 1993; Hanukoglu, 1996). However, the proposed schemes did not cover cytochrome b₅-containing redox pathways, although it has been known that cytochrome b₅ can serve as an effector or electron donor for P450s (Ingelman-Sundberg and Johansson, 1980; Hlavica, 1984; Aoyama et al., 1990; Truan et al., 1993).

Mitochondrial and most of the bacterial P450 systems have three components: an FAD-containing flavoprotein (NADPH or NADH-dependent reductase), an iron-sulphur protein and P450. The eukaryotic microsomal P450 system contains two components: NADPH:P450 reductase (CPR), a flavoprotein containing both FAD and FMN, and P450. CPR appears to be a fusion protein consisting of domains which are homologous to ferredoxin:NADP⁺ reductases (FAD domain) and flavodoxin (FMN domain) (Porter and Kasper, 1986; Smith et al., 1994). A unique prokaryotic two-component P450 monooxygenase system from Streptomyces carbophilus has been described (Serizawa and Matsuoka, 1991). This system is composed of P450sca haemoprotein and NADH-dependent P450 reductase containing both FAD and FMN. Finally, a soluble monooxygenase P450_BM-3 (CYP102) from Bacillus megaterium exists as a single polypeptide chain with two functional parts, the haem and flavin domains (Narhi and Fulco, 1986). It represents a unique bacterial one-component system. However, sequence and functional comparisons show that these domains are more similar to P450 and the flavoprotein of the microsomal two-component P450 monooxygenase system than to the relevant proteins of the three-component system (Ruettinger et al., 1989).

Mammalian nitric oxide synthase (NOS) contains FAD, FMN and haem and was the first eukaryotic catalytically self-sufficient P450-like system described (White and Marletta, 1992). The C-terminal domain of NOS is clearly homologous to CPR, whereas its N-terminal domain is of a low similarity to P450 sequences. The bacterial NOS system, functionally similar to the mammalian one, was recently discovered in Nocardia spp. (Chen and Rosazza, 1994). Analogously, newly described fatty acid hydroxylase from Fusarium oxysporum seems to be a fusion protein of P450 and reductase domains similar to P450_BM-3 (Nakayama and Shoun, 1994). In the context of the proposed classification, mammalian NOS and F. oxysporum monooxygenase could be viewed as eukaryotic one-component P450 systems.

On the other hand, all P450-containing monooxygenase systems described so far share common structural and functional domain architecture (Table I). Here, we mean by `domain' (a segment of) polypeptide existing as an independently folding unit and possessing a certain function. Hence there are no fundamental differences between the protein domain and the individual protein component and all the P450 systems can be considered as three-domain systems (Figure 1):

(i) NADH- or NADPH-dependent FAD-containing reductase (FAD domain), (ii) an iron-sulphur protein (in a three-component system) or FMN-binding domain (in a two- and one-component system) homologous to bacterial flavodoxin, and (iii) P450 protein (haem domain). All the P450s are obviously homologous. In contrast, FAD domains may be categorised into three major families: members of the flavoprotein pyridine nucleotide cytochrome reductases family (a term proposed by Hyde et al., 1991), flavoproteins similar to pyridine nucleotide-disulphide oxidoreductases, and adrenodoxin reductases. Similarly, Fe-S (ferredoxin-like) and FMN-binding domains (flavodoxin-like) are examples of functional analogy. In our opinion, cytochrome b₅ could be categorised as another analogue of this `intermediate' domain. It is worth noting that while these domains possess the same function, they have different specificity. In three-component systems reductase interacts with specific ferredoxin, which, in turn, interacts with the corresponding P450 protein (putidaredoxin reductase -> putidaredoxin -> P450cam, terpredoxin reductase -> terpredoxin -> P450terp, adrenodoxin reductase -> adrenodoxin -> few mitochondrial P450s); it is natural that in one-component systems, at least in vivo, reductase can interact only with the relevant P450 domain of the same polypeptide chain. In a microsomal two-component system, different microsomal P450s interact with a `universal' P450 reductase. Finally, cytochrome b₅ is a non-specific electron donor, so it can interact with P450, microsomal acyl-CoA and sterol desaturases, haemoglobin, cytochrome c, cytochrome b₅, etc. However, it was shown recently that specificity of the interaction of different microsomal P450 proteins from the same organism with cytochrome b₅ exists (Omata et al., 1994a).

Thus, an ancestor of any known P450 monooxygenase system should contain at least three different proteins. An intermediate electron donor component might be presented by one of the functionally interchangeable proteins: flavodoxin-like, ferredoxin or cytochrome b₅. In the case of a flavodoxin-like protein containing system, the fusion of its ancestral gene with the gene encoding the FAD component led to the appearance of new protein family, P450 reductase. Similarly, the fusion of the two ancestral genes, encoding P450 and CPR, resulted in the origin of the natural chimeric P450 system. Hence, P450_BM-3-like enzymes should be considered as the most evolutionarily `advanced' P450 monooxygenase system. Indeed, P450<sub>BM-3</sub> represents the most effective P450 monooxygenase known (Narhi and Fulco, 1986). At the same time, this fusion could result in some lack of flexibility when multiple drug-metabolising enzymes are required. A eukaryotic metabolic system usually employs multiple P450 enzymes of different specificity and one `universal' P450 reductase (for a review, see Hanukoglu, 1992).

FAD domain

(i) FAD flavoprotein/domain shared by members of the ferredoxin:NADP⁺ reductase (FNR) family, also referred to as the flavoprotein pyridine nucleotide cytochrome reductases family (Hyde et al., 1991). This family contains ferredoxin:NADP⁺ reductases, NADH:cytochrome b₅ reductase, NADPH:P450 reductase, NADPH:sulphite reductase (Ostrowski et al., 1989a, b), NADH: and NADPH:nitrate reductases (Campbell and Kinghorn, 1990; Hyde et al., 1991), yeast flavohaemoglobin (Zhu and Riggs, 1992), ß-subunit of phagocyte flavocytochrome b (cytochrome b_-245) (Segal et al., 1992), and phthalate dioxygenase reductase (Correll et al., 1992). In turn, all members of this family seem to share the same structural framework (`FNR-like module'): the N-terminal subdomain, which binds the flavin, and the C-terminal subdomain, which binds pyridine nucleotide (Correll et al., 1993). To date, three-dimensional structures of three members of the family have been solved (see P450 systems in three dimensions). In all of them, the flavin subdomain represents an antiparallel ß-barrel structure, while the NAD(P) subdomain has the ßß topology, typical of pyridine dinucleotide-binding folds (Schulz, 1992). In spite of such structural similarities, the level of amino acid identity between family members is at, or below, the limit of significance (for instance, nitrate reductase is only 15% identical with FNR). It has been suggested that the developed interface between the FAD- and NADPH-binding subdomains in FNR would not allow them to be functionally separated (Karplus et al., 1991). Indeed, experiments on the expression of the FAD-binding subdomain showed that it does not fold correctly in the absence of the NADPH subdomain (Smith et al., 1994).

(ii) Iron-sulphur protein reductases, involved in oxidative metabolism of a variety of hydrocarbons (putidaredoxin reductase, terpredoxin reductase, rubredoxin reductase, ferredoxin:NAD⁺ reductase components of benzene 1,2-dioxygenase, toluene 1,2-dioxygenase, chlorobenzene dioxygenase, biphenyl dioxygenase), share sequence similarity with a number of other flavoprotein oxidoreductases, in particular with the family of pyridine nucleotide-disulphide oxidoreductases (glutathione reductase, trypanothione reductase, lipoamide dehydrogenase, mercuric reductase, thioredoxin reductase, alkyl hydroperoxide reductase), NADH oxidase, NADH peroxidase and flavoprotein subunit of flavocytochrome c sulphide dehydrogenase (Eggink et al., 1990; Kuriyan et al., 1991; Mason and Cammack, 1992). Comparison of the crystal structures of human glutathione reductase and Escherichia coli thioredoxin reductase reveals different locations of their active sites, suggesting that the enzymes diverged from an ancestral FAD/NAD(P)H reductase and acquired their disulphide reductase activities independently (Kuriyan et al., 1991). Since glutathione reductase (GR) represents the structural prototype of the family, we shall refer to this large group of proteins as the GR family. All crystal structures resolved so far for the members of this family show similar topology, but the relative orientations of their FAD- and NAD(P)H-binding subdomains may vary significantly (see P450 systems in three dimensions). By contrast with the FNR family, the FAD- and NAD(P)H-binding subdomains in the GR family share both the sequence and spatial similarity (ßß fold), suggesting that these proteins evolved by gene duplication (Schulz, 1980).

(iii) NADPH:adrenodoxin reductase, which shows no sequence similarity with other known ferredoxin reductases over the entire length of sequence (Hanukoglu and Gutfinger, 1989). However, the FAD and NAD(P)-binding sites appear in both putidaredoxin reductase and adrenodoxin reductase at nearly identical positions (Hanukoglu, 1996). Furthermore, adrenodoxin reductase shares local similarity with glutamate synthases and NADH peroxidase.

Ferredoxins

Flavodoxins

Cytochromes b5

Cytochrome b₅ is homologous to the haem-containing redox domain of a number of oxidoreductases, such as plant and fungal nitrate reductases, chicken sulphite oxidase and yeast flavocytochrome b₂ (L-lactate dehydrogenase). On the basis of sequence comparison, it was also hypothesised that the globin family was evolved from an ancestral cytochrome b₅-like protein (Runnegar, 1984). It is notable that all cytochromes b₅ and their homologues described so far were found only in eukaryota.

P450 domain

The same is true for other domains of the system: the molecular evolution of each component does not follow phylogeny in general, although phylogenetic analysis can be applied to several groups of orthologous proteins (most eukaryotic NADPH:P450 reductases, microsomal cytochromes b₅ and NADH:cytochrome b₅ reductases).

Taking into account that horizontal gene transfer events may occur, the subdivision of P450-containing monooxygenase systems into prokaryotic and eukaryotic seems to be quite formal. Furthermore, there are no fundamental differences between P450 systems of various cellular localisation. For instance, mitochondrial P450 could be easily converted into a microsomal system solely by altering the targeting signal sequence. Experiments with modified mitochondrial P450c27 (CYP27), whose mitochondrial targeting signal sequence was replaced by the microsomal targeting signal of microsomal P450, proved that this protein, when expressed in yeast, is localised on microsomes and shows monooxygenase activity in the presence of adrenodoxin and NADPH:adrenodoxin reductase (Sakaki et al., 1992).

Despite the apparent simplicity of the proposed model, the evolutionary relationships of prokaryotic and eukaryotic one-, two-, and three-component P450-containing systems are not obvious. Indeed, it is known that, on the basis of sequence similarity, all P450s can be categorised into two main classes: B-class and E-class (Gotoh, 1993). Only P450 proteins of prokaryotic three-component systems and fungal P450nor (CYP55) belong to B-class P450s, whereas all other known P450s from distinct systems are of E-class. Moreover, the data suggest that the divergence of the P450 superfamily into B- and E-classes, and within E-class, further divergence into stable P450 groups should be very ancient and occured before the appearance of eukaryotes. Therefore, the origin of membrane anchors should be an independent event, at least in each major P450 group. A very weak similarity between the N-terminal membranous segments could be explained as a result of the `evolutionary game' with the absence of charged residues (or, at least, of uncompensated charges). On the other hand, the acquisition of membrane-binding properties by one of the protein components of the microsomal monooxygenase system resulted in anchoring other components to the membrane, although this can occur in different ways. Microsomal P450 proteins and CPRs are anchored by the N-terminal transmembrane segment, whereas microsomal cytochromes b₅ have a C-terminal hydrophobic segment. Other components can possess lipid anchors such as NADH:cytochrome b₅ reductase (Ozols et al., 1984) and endothelial NOS (Busconi and Michel, 1993), which have an N-terminal myristoylated residue.

The P450 molecule represents an /ß protein which is shaped like a triangular prism; the overall structure might be roughly divided into an `-rich domain' (`right side') and a `ß-rich domain' (`left side') (Cupp-Vickery and Poulos, 1995). However, this division appears to be artificial since `-rich' and `ß-rich domains' comprise the discontinuous assemblies of secondary structure segments and do not constitute independent folding units. Although the sequence identity between any two P450s with known 3-D structures reaches only 20% or less, the overall topology of these proteins is similar (Figure 2a and b). However, P450s differ in the orientation of several helices. The root mean square (r.m.s.) deviation of C atoms in helical segments of P450 is 2.8 Å (for comparison, FNR and PDR share 15% identity and the r.m.s. deviation of C atoms for FNR-like core is only 1.5 Å). The most dramatic variations between the P450 structures are found in regions responsible for substrate binding and access (Li and Poulos, 1994). In the crystal structure of P450_BM-3, the access channel for the substrate, a long chain fatty acid, is open, whereas in P450cam it is closed (Li and Poulos, 1995). The B' helix, which is responsible for substrate binding and hydroxylation specificity in P450cam, is positioned differently in other P450s. Although the crystal structure of P450cam has been extensively used to predict 3-D structures for eukaryotic P450 (Ferenczy and Morris, 1989; Graham-Lorence et al., 1991; Poulos, 1991; Zvelebil et al., 1991; Vijayakumar and Salerno, 1992; Boscott and Grant, 1994), it is worth noting that even the prediction of 3-D structure for other bacterial enzymes using P450cam as a template apparently cannot be successful with current homology modelling techniques. The recent studies of Ruan et al. (1994) revealed that P450_BM-3 provides a more suitable template for homology modelling of thromboxane synthase (CYP5) and possibly a number of other P450 families. However, keeping in mind the known structural differences between B-class P450s, the models (especially of substrate-binding sites) based on the sole structure of the E-class P450 representative, should be treated with caution. Information on other 3-D structures of this large group would obviously be of primary interest for homology modelling.

FNR-like proteins consist of ß-barrel (FAD) and pyridine nucleotide-binding (NADPH) subdomains with well conserved topology (Figure 2g). However, they also show some important structural differences. Minor differences in the structure of the ß-barrel subdomain of PDR and FNR result in selectivity between FMN and FAD (Correll et al., 1993). Comparison of FAD complexes of FNR and nitrate reductase revealed that even the same cofactor differs in orientation of its adenine-ribose part (Lu et al., 1994). The specificity of PDR and nitrate reductase for NAD(H) and FNR for NADP(H) is presumed to be determined by the presence of either an acidic or basic residue, respectively, close to the 2'-phosphate binding site. The relative orientation of two subdomains in these flavoproteins also varies substantially, reflecting different rotations around a common axis.

The members of the glutathione reductase family, although having a similar overall topology, show a variety of structural differences concerning packing of subdomains, mode of pyridine nucleotide binding and localisation of redox-active cysteine residues. A lack of the interface subdomain in the case of thioredoxin reductase (Kuriyan et al., 1991) and the appearance of an extra N-terminal domain in the case of mercuric reductase (Schiering et al., 1991) are the most impressive examples of such differences. Thus, in spite of an apparent abundance of structural information (seven crystal structures for this family), the 3-D database is still very restricted for modelling reductase components of bacterial P450-containing systems.

To date, no X-ray or NMR structure is known for complexes of the redox domains reviewed here. The only resolved example of an interface between plant-type Fe₂S₂ ferredoxin and FNR-like domains is PDR, where this interface is formed within a single polypeptyde. The contacts between these two domains include eight direct hydrogen bonds and six solvent bridges (Correll et al., 1993). It is tempting to suggest PDR as a guide for construction of the models for complexes of FNR with other electron carriers; however, the model FNR-ferredoxin complex that was built in that way was not consistent with the results of experiments. Furthermore, plant-type Fe₂S₂ ferredoxins do not share homology with any domain group of known P450-containing systems. In this connection, the recent crystallisation and preliminary X-ray studies of CPR (Djordjevic et al., 1995) cannot be overrated. Among other things, this long-awaited structure will allow an understanding of spatial relationships between redox domains.

Although 3-D structures are available for two interacting in vivo proteins, P450cam and putidaredoxin, it is still unknown which structures form the interface between them. Experiments by Stayton et al. (1989) have suggested a putative binding site for putidaredoxin based on competition studies between putidaredoxin and cytochrome b₅ in their association with P450cam. Computer-aided docking of two 3-D structures has been used to build a model of a cytochrome b₅-P450cam complex, where the interface is formed by carboxyl groups of cytochrome b₅ and basic residues of P450cam (Stayton et al., 1989; Stayton and Sligar, 1990; Omata et al., 1994a, b). By analogy, it was hypothesised that putidaredoxin contains a cluster of acidic residues which interact with P450cam. On the other hand, it has been proposed that solvent-exposed side chains of several hydrophobic residues of putidaredoxin (Trp106, Val4, Val6 and Val98) are involved in the binding of P450cam (Pochapsky et al., 1994).

A model of the cytochrome b domain of nitrate reductase, built on the basis of bovine cytochrome b₅, was docked to the FAD (FNR-like) domain of nitrate reductase (Lu et al., 1995). It was shown that the surface of cytochrome b₅ can be docked on that of reductase in a number of different orientations, one of which is energetically most favourable.

Nitric oxide reductase: homologous but not analogous

Interestingly, a functional analogue of P450nor, the cytochrome b subunit of bacterial NO reductase (NorB), is homologous to subunit I of cytochrome oxidase. It was proposed that cytochrome oxidase developed from NO reductase (Saraste and Castresana, 1994), particularly because NO was present in the earth's atmosphere before the appearance of molecular oxygen. Despite the fact that P450 and cytochrome oxidase are not homologous, both represent part of redox chains that should evolve from an anaerobic to an aerobic reactions catalysis. We hypothesised earlier that P450 might be one of the most ancient respiratory enzymes (Degtyarenko and Archakov, 1993). Perhaps P450nor, which has retained a nitrate respiration function, can serve as evidence of this early period of P450 evolution.

It was originally proposed that a fungal genome acquired CYP55 gene via horizontal gene transfer from bacteria (e.g. streptomycetes), and then divergent evolution led to a novel function of P450, distinct from monooxygenase (Kizawa et al., 1991). However, the scheme of P450nor evolution should be different, assuming that monooxygenase activity is not a primary function of P450s! A redox chain as complex as a three-component (or three-domain) P450 system could not arise from scratch; obviously, there must be some self-sufficient P450-like haemoprotein somewhere among its ancestors. Thus, it is more plausible that both Streptomyces and Fusarium obtained P450 via horizontal gene transfer from some denitrifying bacterium. To activate the P450 monooxygenase ability, it was necessary to use two additional protein components (note that none of these are unique to P450 systems only). It is of further interest that F. oxysporum also possesses P450 monooxygenase, microsomal fatty acid hydroxylase resembling P450_BM-3 (Nakayama and Shoun, 1994). Thus, anaerobic and aerobic P450 redox chains can coexist within one cell.

The pronounced sequence similarity of NOS with CPR (Bredt et al., 1991) and spectral properties typical for P450 haemoproteins suggested that NOS is a eukaryotic one-component P450 system. Reactions catalysed by NOS, such as L-arginine N-hydroxylase, N-hydroxy-L-arginine monooxygenase and NADPH:cytochrome c reductase, prove the functional identity of haem and reductase domains of NOS with P450 and of CPR, respectively. On the other hand, if homology of NOS and reductase is more or less obvious, there are different views on the origin of the similarity between the NOS haem domain and P450s. Nelson et al. (1993) classified NOS as an example of convergent evolution. Renaud et al. (1993) showed the functional identity of NOS and P450 3A as hydroxy-L-arginine monooxygenases and proposed that a nine amino acid long peptide, highly conserved among all NOS proteins and similar to the haem-binding consensus sequence of P450s, should be responsible for haem binding in NOSs. It should be noted that the relative location of these segments in protein sequences differs substantially, which causes serious problems in the alignment of P450 and NOS sequences. Alternatively, in our previous work (Degtyarenko and Archakov, 1993), employing alignment of N-terminal domains of P450_BM-3 and NOS, it was demonstrated that the similarity between these sequences can be observed without introducing long gaps. However, the segment containing conserved cysteine residue of NOS, which may correspond to the haem ligand of P450, was found to be completely different from the well known P450 `haem cystein' signature.

Besides the NOS family, only P450_BM-3 represents a one-component P450 system with a known sequence. Even assuming common ancestry of their haem domains, this does not mean that these systems are descended from a common P450/CPR fusion protein. Taking into account that the reductase domain is much more conserved than the haem domain, it is more likely that corresponding gene fusion events occured independently in NOS and CYP102 families.

Separated haem (P450) and flavin (CPR) domains obtained by proteolysis (Narhi and Fulco, 1986) or genetic engineering (Li et al., 1991; Munro et al., 1994) were used in studies of kinetics of electron transfer and catalysis. Each domain was shown to retain characteristic catalytic and spectral properties, whereas the rate of electron transfer between the isolated domains is considerably slower than for the native enzyme. It was proposed that the proper mutual orientation of P450 and reductase domains in the intact P450_BM-3 protein is maintained by the linker region (Munro et al., 1994). Analogously, experiments on limited proteolysis revealed that the haem and flavin domains of neuronal NOS can be isolated in functionally intact forms (Sheta et al., 1994).

Smith et al. (1994) demonstrated that the FAD and FMN domains of human CPR, expressed in E. coli as independent polypeptides, could be reconstituted to form a functional complex possessing P450 and cytochrome c reductase activity. Thus, an artificial system simulating a possible ancestral three-component P450 system was constructed. The catalytically active FAD domain of corn leaf NADH:nitrate reductase was expressed in E. coli and was found to exhibit spectral properties identical with NADH:cytochrome b₅ reductase (Hyde and Campbell, 1990). Similarly, genetically engineered cytochrome b₅-like domains of Chlorella vulgaris NADH:nitrate reductase (Cannons et al., 1991) and yeast flavocytochrome b₂ (Brunt et al., 1992), showing spectra virtually identical with those of microsomal cytochrome b₅, were expressed independently in E. coli.

Reciprocally, the possibility of obtaining a functional one-component P450 system from a two-component system has been shown in gene fusion experiments. To date, the following eukaryotic analogues of P450_BM-3 have been constructed: bovine CYP17/yeast CPR (Shibata et al., 1990), rat CYP1A1/yeast CPR (Sakaki et al., 1994) and human CYP1A1/human CPR (Wittekindt et al., 1995) expressed in Saccharomyces cerevisiae; bovine CYP17/rat CPR, rat CYP4A1/rat CPR (Fisher et al., 1992; Shet et al., 1994) and human CYP3A4/human or rat CPR (Shet et al., 1993) expressed in E. coli.

Two other domains considered in this review were recently used by Quinn et al. (1994), who constructed and expressed a functional chimeric protein comprising the soluble domain of rat cytochrome b₅ as N-terminus and the FAD domain of spinach assimilatory NADH:nitrate reductase as C-terminus. This chimera was termed flavocytochrome b₅ by analogy with the yeast flavocytochrome b₂; note, however, that the flavin domain of flavocytochrome b₂ is not homologous with the FAD domain of the FNR family, but represents an eightfold ß/-barrel (Lindqvist et al., 1991) and is shared by a large number of different enzymes (for a review, see Farber and Petsko, 1990).

Pueyo et al. (1989) obtained a covalent complex between Azotobacter vinelandii flavodoxin and Anabaena ferredoxin:NADP⁺ reductase; similarly, Pirola et al. (1994) constructed a cross-linked Desulfovibrio vulgaris flavodoxin-spinach FNR complex. Both systems possess NADPH:cytochrome c reductase activity. In fact, these functional heterologous complexes represent novel CPRs.

Finally, P450 systems without other protein components can be briefly discussed. We have already mentioned the self-sufficient system P450nor from F. oxysporum catalysing NADH-dependent NO reduction. Recently, the monooxygenase activity of `monodomain' P450 systems has also been shown. Gonvindaraj et al. (1994) found that, in the presence of FMN and NADPH, the haem domain of P450_BM-3 can catalyse, at a slow rate, the hydroxylation of a fatty acid substrate (myristic acid) without the reductase domain. A similar reaction has been shown for microsomal P450 (rabbit CYP2B4)-catalysed p-hydroxylation of aniline, in the presence of 1000-fold excess of FMN over P450 (Uvarov et al., 1994). At the same time, it was shown that the covalent binding of FMN with CYP2B4 using carbodiimide yielded a conjugate able to catalyse NADPH-dependent N-demethylation of aminopyrine and dimethylaniline and p-hydroxylation of aniline, at rates which are only 30-40% lower than those of the microsomal system.

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Received March 16, 1995; revised June 8, 1995; accepted June 21, 1995

Fig. 1. Schematic representation of the domain structure of P450 systems (GIF ).

Fig. 2. Three-dimensional structures of redox domain representatives.

(a) B-class P450: Pseudomonas putida P450cam (CYP101), 2CPP (Poulos et al., 1987)
PDF	POSTSCRIPT	GIF
(b) E-class P450: haem domain of Bacillus megaterium P450BM-3 (CYP102), 2HPD (Ravichandran et al., 1993)
PDF	POSTSCRIPT	GIF
(c) Fe2S2 ferredoxins: P. putida putidaredoxin, 1PUT (Pochapsky et al., 1994)
PDF	POSTSCRIPT	GIF
(d) Fe4S4 ferredoxins: Desulfovibrio africanus ferredoxin, 1FXR (Sery et al., 1994)
PDF	POSTSCRIPT	GIF
(e) cytochromes b5: bovine cytochrome b5, 3B5C (Argos and Mathews, 1975)
PDF	POSTSCRIPT	GIF
(f) FMN domain: Desulfovibrio vulgaris flavodoxin, 2FX2 (Watt et al., 1991)
PDF	POSTSCRIPT	GIF
(g) FAD domain (FNR-like): pig NADH:cytochrome b5 reductase, 1NDH (Nishida et al., 1995)
PDF	POSTSCRIPT	GIF
(h) FAD domain (GR-like): Escherichia coli thioredoxin reductase, 1TRB (Kuriyan et al., 1991)
PDF	POSTSCRIPT	GIF

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