Diverse role of conserved aromatic
amino acids in the electron transfer of cytochrome P450 catalytic
functions: site-directed mutagenesis studies
December 20, 1996
Key Words: Cytochrome P450, Electron
transfer, Site-directed mutagenesis, Aromatic amino acid.
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.
(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).
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).
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.
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
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.