Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0 Å resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B helix, ␤ sheet 4, and part of ␤ sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds. The cytochromes P4504 constitute a superfamily of heme-containing enzymes that catalyze the metabolism of a wide variety of endogenous and xenobiotic compounds. This is accomplished through the activation of molecular oxygen by the heme group, a process that involves the delivery of two electrons to the P450 system followed by cleavage of the dioxygen bond, yielding water and an activated iron-oxygen species (Compound 1), which reacts with substrates through a variety of mechanisms (1). In eukaryotic species, the electron source is a single flavoprotein, the FAD/FMN-containing cytochrome P450 reductase, which binds to the largely basic proximal face of the cytochrome through a number of salt bridges. Of the known human isoforms, cytochrome P450 2D6 is responsible for the metabolism of at least 20% of known drugs (2), with only 3A4 being responsible for a higher (50%) percentage.The cDNA encoding human P450 2D6 has been characterized (3) and subsequently localized to chromosome 22 in the q13.1 region (4). A relatively large number of genetic polymorphisms have been described for 2D6, some of which can either result in rapid or very poor metabolism. One well characterized allelic variant is responsible for a condition known as debrisoquine/sparteine type polymorphism (5, 6). This arises as a result of various genetic mutations and affects a significant percentage of the Caucasian population (7). It results in the defective metabolism of a number of important drug molecules, including debrisoquine, from which the condition got its name. The inability of patients to turn over compounds such as debrisoquine eventually leads to toxic levels of the drug in t...

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

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Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.

Site‐directed mutants of the pea plastocyanin gene in which the codon for the surface‐exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild‐type plastocyanin by 1H‐NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped‐flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8‐fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40‐fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.

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The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.