Diverse role of conserved aromatic amino acids in the electron transfer of cytochrome P450 catalytic functions: site-directed mutagenesis studies

Toru Shimizu

December 20, 1996

Key Words: Cytochrome P450, Electron transfer, Site-directed mutagenesis, Aromatic amino acid.

Abstract

Cytochrome P450 accepts electrons from electron transfer proteins to make monooxidation reactions proceed. It was suggested that aromatic amino acids in the P450 molecule are involved in intramolecular or intermolecular electron transfer reaction . Hitherto, more than ten conserved aromatic amino acids have been replaced with aliphatic or other aromatic amino acids by referring to the previous suggestion. However, obtained catalytic activities of the aromatic mutants of various P450s are inconsistent with each other. We briefly review the role of the aromatic amino acids in the intramolecular or intermolecular electron transfer reactions of P450s based on the previously obtained results by us together with those obtained in other laboratories.

Introduction

Cytochrome P450 (P450) is a hemoprotein which catalyzes monooxidation reactions of external and endogenous compounds by using molecular oxygen and electrons from NADPH or NADH through electron transfer proteins (Guengerich, 1991, Porter & Coon, 1991, Ortiz de Montellano, 1995). Perhaps basic amino acids of the heme proximal surface of the P450 proteins are engaged in the interaction with counter ionic amino acids of the electron-transfer proteins (Shimizu et al., 1991c, Mayuzumi et al., 1993). However, it is not clear whether or not electron(s) transfer via the ionic bridges between the redox complexes. Also, it is not clear which route electrons take to reach the heme iron after they come into contact with the P450 molecular surface. If the 1st electron should enter at the heme proximal site of the P450 molecular surface (Nakano et a., 1995, 1996a), how can the 2nd electron gain access to the heme active site of P450? It is possible that electron transfer between two redox partners may involve a pathway(s) that includes overlapping p orbitals of aromatic amino acid residues (Mayuzumi et al., 1994). The aims of this review are to summarize briefly our current knowledge of the electron transfer of the P450 system and to describe recent progress in this area where much still remains to be learned.

Proximal Phe Interacting the Heme

Phe449 of P450 1A2 is nearly completely conserved in both bacterial and eukaryotic P450s (Degtyarenko,1996; Nelson and Strobel, 1988) (Figure 1). This highly conserved residue is situated on the proximal surface and is in close proximity to the heme place with the distance about 3.5 * in bacterial P450cam (Figure 2) (Poulos et al., 1985, 1987). It was hypothesized that this aromatic residue participates in electron transfer to the buried heme group from the redox partner (Sligar et al., 1991). Mutations at this Phe in both bacterial and eukaryotic P450s often resulted in the heme loss from the enzyme, thus it is experimentally shown that this Phe is actually in close proximity to the heme plane (Table I) (Sligar et al., 1991; Porter, 1994, Shimizu et al., 1988). Nevertheless, mutants at this position were made for P450 1A2, P450 101, and P450 2E1 (Shimizu et al., 1988, Porter, 1994, Yasukochi et al., 1994). Catalytic activities of P450 1A2 Phe449 mutants, in which aliphatic amino acids were replaced, were 13% - 30% of that of the wild type (Table I). P450 101 Phe350Leu mutants showed 66% of the wild type. In addition, rate constants for the reduction of Phe350His, Phe350Tyr and Phe350Leu mutants of P450 101 by reduced putidaredoxin, a redox partner, were comparable to that of the wild type (Yasukochi et al., 1994). Therefore, if the mutants keep the heme properly oriented in the active site, the mutants showed catalytic activities enough to suggest that this conserved Phe aromaticity is not crucial in the catalytic functions of P450s. In eukaryotic P450s, this conserved Phe is likely to be a little more important than that of bacterial P450, since activities of P450 1A2 are lower, if not remarkably, than the wild type (Table I). Structure of membrane-bound eukaryotic P450s may be subtly different from that of water-soluble bacterial P450. Difference of the structure of redox partners, non-heme protein putidaredoxin for P450 101, and flavine proteins for eukaryotic P450s, may also explain the activity difference of Phe mutants between P450 1A2 and P450 101. P450 2E1 Phe429Trp and Phe429Tyr mutants showed catalytic activities comparable to those of the wild type (Table I). However, the aromaticity at this position is still kept in the mutants and thus it is unlikely to prove or disapprove that the Phe429 aromaticity significantly contributes to the electron transfer and/or catalytic function in the P450 2E1 mutants (Porter, 1994). In summary, it is possible that this conserved Phe play a role in keeping the heme plane at the active site with hydrophobic p-p interaction and/or in establishing the redox potential of the heme iron.

Trp at the Heme Proximal Site

There is a conserved Trp in the amino-terminal site of the eukaryotic P450 molecule (Degtyarenko, 1996; Nelson and Strobel, 1988) (Figure 1). This aromatic amino acid residue seemed likely to play a significant role in the electron transfer of eukaryotic P450s (Nelson and Strobel, 1988). Baldwin et al. (1991) proposed a "covalent switching" mechanism to explain the direct involvement of this conserved Trp of P450s and putidaredoxin Trp as a mediater of the electron-transfer flow between redox partners in P450 systems (Figure 2). In fact, mutations at this conserved Trp position of P450s tend to lose the heme from the active site of the enzyme, or change the heme spin state (Munro et al., 1994, Shimizu et al., 1996). Therefore, it seems likely that this Trp somehow interacts with the heme and contributes to the heme binding to the P450 apoprotein. Nevertheless, several mutants at this position were made for P450 BM3, P450 1A2 and P450 2C2. In contrast to the previous suggestion, Trp97Phe, Trp97Tyr and Trp97Ala mutants of P450 BM3 retained enough catalytic activities (Table II) (Munro et al., 1994). Also, catalytic activity of Trp132Leu mutant of P450 1A2 was 30-40% of the wild type (Shimizu et al., 1996). These results suggest the equivocal role of this Trp in the catalytic function of P450s. On the other hand, Trp120Ala, Trp120Leu, Trp120Ile, and Trp120Pro mutants of P450 2C2 showed less than 25 % catalytic activity of the wild type (Straub et al., 1993). Unfortunately, however, absorption spectra were not obtained for the expressed P450 2C2 mutants. Thus, the work did not obtain any information about the absorption spectra of the mutants. It is, therefore, not clear if the heme is actually bound to the active site of the mutants. Thus, the catalytic activities described for the several Trp120 mutants of P450 2C2 (Straub et al., 1993) require further studies to clarify this finding.

This conserved Trp is not found in P450 101, but putidaredoxin, an electron-transfer protein for P450 101, has Trp in the carboxyl-terminal site. Mutations of Trp of putidaredoxin caused low catalytic activities (Davies et al., 1990). However, it was suggested that the presence of carboxyl-terminal aromatic residue is required for relatively high P450 101 affinity of the reduced form relative to the oxidized form of putidaredoxin. Namely, detailed kinetic studies showed that decreased catalytic rates with a non-aromatic substituted mutant result from less efficient association with P450 101, and not from deficient electron transfer to the redox partner as shown in Table III (Davies and Sligar, 1992). NMR studies also suggest that the Trp of putidaredoxin is important for the binding to P450 101 (Pochapsky et al., 1994).

Together, it seems most likely that the conserved Trp is not directly engaged in the intermolecular or intramolecular electron transfer reaction in the P450 systems in contrast to the proposed mechanism by Baldwin et al. (1991). The conserved Trp may serve an important role 1) in the heme binding to the active site; 2) in formation of the tertiary structure of eukaryotic P450 (Munro et al., 1994) or 3) in keeping the appropriate redox potential of the heme iron.

Other Aromatic Amino Acids

There are several conserved and non-conserved aromatic amino acids in P450s (Figure 1). Mutations at those sites of P450 1A2 did not lose the heme from the active site in terms of the CO-reduced absorption spectra (Table IV) (Shimizu et al., 1996). His163 seems important to keep an appropriate redox potential of P450 1A2 for optimum electron transfer to occur from cytochrome b5 (Mayuzumi et al., 1994). However, most of the catalytic activities of yeast microsomes containing the aromatic mutant enzymes described were comparable to the wild type (Table I). At least on the basis of catalytic activity of the non-aromatic amino-acid-substituted mutants, it can be conjectured that those aromatic amino acids are not directly implicated in the electron transfer of the eukaryotic P450 function. It seems that these conserved aromatic amino acid residues are important to keep the P450 protein three-dimensional structure or form hydrophobic substrate-binding sites (Ortiz de Montellano, 1995).

Summary

Electron transfer reactions of microsomal P450s are rather slow with rates of the sec order (Shimizu et al., 1991c). Thus, it is possible that electrons slowly flow in the P450 protein through various peptides, aromatic amino acid residue or some other molecular route. This is in contrast to fast electron transfer reaction having msec or nsec order rate according to the "electron tunnelling pathway" (Beratan et al., 1991) or "quantum biomechanics" (Dutton and Mosser, 1994). However, the distance or thickness between the proximal protein surface and the heme is ca. 8 * based on the P450 101 crystal structure (Poulos et al., 1985). This range is rather thin and within the rage 10-25 * of electron tunnelling or quantum biomechanics (Beratan et al., 1991; Dutton and Mosser, 1994; Dryhurst and Niki, 1988; Evenson and Karplus, 1993; Pelletier and Kraut, 1992; Zhou et al., 1995). Floppy through-space contacts are suggested to be more important than p-electron pathways for the intramolecular electron transfer according to electron tunnelling pathways mechanism (Beratan et al., 1991). Hydrogen bonds and reorganization energies of peptides are also suggested to be more important than electron tunnelling according to quantum biomechanims theory (Dutton and Mosser, 1994). According to those mechanisms, p-electrons of aromatic amino acids are not required for intramolecular electron transfer after electrons contact the protein surface. Thus, it is possible that electrons skip to the heme active site from the protein surface without passing through any specific chemical bonds. As a matter of fact, the P450 heme easily accepts electrons from photo-reduced dyes that perhaps bind to P450 protein surface site(s) different from the reductase (Nakano et al., 1995, 1996). Together, it seems most likely that the aromatic amino acid residues are not essential for the intra- and intermolecular electron transfer reaction of the P450 system. The aromatic residue may instead contribute to form the protein three-dimensional structure using its hydrophobic character and/or to enhancing the electron transfer efficiency. Distal amino-acids role in the P450 catalytic function associated with the heme redox state and the electron transfer should also be reminded here (Nakano et al., 1996b; Shimizu et al., 1991a,b,1994). Incidentally, electron transfer reactions of the photosynthetic system are very fast with rates much less than msec (Dutton and Mosser, 1994). Electrons quickly run over a wide range of distance, 10 - 20 *, without passing through any specific polypeptide or aromatic amino acid residue in the photosynthetic bacterial system (Dutton and Mosser, 1994). Further study will be required to clarify this issue in regard to the inter- and intramolecular electron transfer of the P450 system by comparing the P450 electron transfer reaction with the photosynthetic system.

Acknowledgements

This work was supported in part by a Grant from Sumitomo Science Foundation and Grants-in-Aid (7680670, 7558083) from the Ministry of Education, Science, Sports and Culture of Japan.

References

Baldwin, J. E., Morris, G. M., and Richards, W. G. (1991) Proc. Royal Soc. London B, 245, 43-51.

Beratan, D. N., Onuchic, J. N. and Gray, H. B. (1991) Metal Ions in Biological Systems, 27, 97-127.

Davies, M. D., Qin, L., Beck, J. L., Suslick, K. S., Koga, H., Horiuchi, T. and Sligar, S. G. (1990) J. Amer. Chem. Soc., 112, 7396-7398.

Davies, M. D. and Sligar, S. G. (1992) Biochemistry, 31, 11383-11389.

Degtyarenko, K. N. (1996) Internet at URL: http://bioinf.leeds.ac.uk/cgi-bin/prints.sh?d%2Ffu+code+%22P450%2 )

Dryhurst, G. and Niki, K. Eds. (1988) Redox Chemistry and Interfacial Behavior of Biological Molecule, Plenum Press, New York.

Dutton, P. L. and Mosser, C. C. (1994) Proc. Natl. Acad. Sci. USA, 91, 10247-10250.

Evenson, J. W. and Karplus, M. (1993) Science, 262, 1247-1249.

Furuya, H., Shimizu, T., Hirano, K., Hatano, M., Fujii-Kuriyama, Y., Raag, R. and Poulos, T. L. (1989a) Biochemistry, 28, 6848-6857.

Furuya, H., Shimizu, T., Hatano, M. and Fujii-Kuriyama, Y. (1989b) Biochem. Biophys. Res. Commun., 160, 669-676.

Guengerich, F. P. (1991) J. Biol. Chem., 266, 10019-10022.

Mayuzumi, H., Sambongi, C., Hiroya, K., Shimizu, T., Tateishi, T. and Hatano, M. (1993) Biochemistry, 32, 5622-5628.

Mayuzumi, H., Shimizu, T., Sambongi, C., Hiroya, K. and Hatano, M. (1994) Arch. Biochem. Biophys., 310, 367-372.

Munro, A. W., Malarkey, K., McKnight, J., Thompson, A. J., Kelly, S. M., Price, N. C., Lindsay, J. G., Coggins, J. R. and Miles, J. S. (1994) Biochem. J., 303, 423-428.

Nakano, R., Konami, H., Sato, H., Ito, O. and Shimizu, T. (1995) Biochim. Biophys. Acta, 1252, 245-250.

Nakano, R., Sato, H. and Shimizu, T. (1996a) J. Photochem. Photobiol. B: Biol., 32, 171-176

Nakano, R., Sato, H., Watanabe, A., Ito, O. and Shimizu, T. (1996b) J. Biol. Chem., 271, 8570-8574

Nelson, D. R. and Strobel, H. W. (1988) J. Biol. Chem., 263, 6038-6050. Internet at URL: http://drnelson.utmem.edu/p450apub192.html

Ortiz de Montellano, P. R. Ed. (1995) Cytochrome P-450, Structure, Mechanism and Biochemistry, 2nd Ed. Plenum Press, New York.

Pelletier, H. and Kraut, J. (1992) Science, 258, 1748-1755.

Pochapsky, T. C., Ratnaswamy, G. and Patera, A. (1994) Biochemistry, 33, 6433-6441.

Porter, T. D. (1994) Biochemistry, 33, 5942-5946.

Porter, T. D. and Coon, M. J. (1991) J. Biol. Chem., 266, 13469-13472.

Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C. and Kraut, J. (1985) J. Biol. Chem., 260, 16122-16130.

Poulos, T. L., Finzel, B. C. and Howard, A. J. (1987) J. Mol. Biol., 195, 687-700.

Shimizu, T., Hirano, K., Takahashi, M., Hatano, M. and Fujii-Kuriyama, Y. (1988) Biochemistry, 27, 4138-4141.

Shimizu, T., Ito, O., Hatano, M. and Fujii-Kuriyama, Y. (1991a) Biochemistry, 30, 4659-4662.

Shimizu, T., Sadeque, A. J. M., Sadeque, G. N., Hatano, M. and Fujii-Kuriyama, Y. (1991b) Biochemistry, 30, 1490-1496.

Shimizu, T., Tateishi, T., Hatano, M. and Fujii-Kuriyama, Y. (1991c) J. Biol. Chem., 266, 3372-3375.

Shimizu, T., Murakami, Y. and Hatano, M. (1994) J. Biol. Chem., 269, 13296-13304.

Shimizu, T., Tateishi, T. and Mayuzumi, H. (1996) unpublished results.

Sligar, S. G., Filipovic, D. and Stayton, P. S. (1991) Methods in Enzymol., 206, 31-49.

Straub, P., Ramarao, M. K. and Kemper, B. (1993) Biochem. Biophys. Res. Commun., 197, 433-439.

Yasukochi, T., Okada, O., Hara, T., Sagara, Y., Sekimizu, K. and Horiuchi, T. (1994) Biochim. Biophys. Acta, 1204, 84-90.

Zhou, J. S., Nocek, J. M., DeVan, M. L. and Hoffman, B. H. (1995) Science, 269, 204-207.