Minireview

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 16, Issue of June 5, pp. 10019-10022,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

the host andmay cause toxicity or cancer (10). The other major hypothesis, that of the importance of P-450s in normalmetabolism, also has validity. Even inyeasts, lanosterol a key P-450 activity (11). In mammals many 14a-demethylation is steroidogenic tissues contain critical P-450s. The lack of funcF. Peter Guengerich tional P-450 21A2 (catalyzing 21-hydroxylation of progesterone From the Departmentof Biochemistry and Center in and pregnenolone) constitutes a serious congenital disease (12), Molecular Toxicology, Vanderbilt University School of and P-450s are also known to play important roles in vitamin D Medicine, Nashville, Tennessee 37232-0146 homeostasis (13). Most of the eukaryotic P-450s have been considered to be located in the endoplasmic reticulum (i.e. microsoCytochrome P-450 enzymes (EC 1.14.14.1, nonspecific mon- mal), except for the two P-450 11products in steroidogenic tisuses ooxygenase) catalyze the oxidations of many chemicals (1).The (14). These mitochondrialP-450s and their bacterial counterparts mass of the substrates ranges from that of ethylene (Mr 28) to utilize electron transport systems consisting of a flavoprotein and that of cyclosporin A ( M , 1201). The classification of a hemopro- an iron-sulfur protein (adrenodoxin),, instead of the single flavotein asa P-450 is defined by its absorption spectrum; the Fe(I1)- protein (containing FMN and FAD) in the endoplasmic reticuCO complex has a characteristicabsorptionmaximum(Soret lum. Further, the mitochondrial and bacterial P-450s are usually band) near 450 nm due to axial ligation with a cysteine thiolate much more selective in termsof the range of substrates eachwill of the protein (withor without substrate present). This cysteine oxidize. In recent years Avadhani and others (13, 15) have found residue is present ina relatively well conserved region, -80% into evidence that some hepatic P-450s are found in mitochondria and the proteinfrom the N terminus. Collectively there are thousands have significant catalytic activities toward xenobiotics as well as of potential substrates for the P-450s; each of the P-450s may steroids (13, 15). have a rather strict limitationof catalytic specificity (e.g. P-450s What do we know about thesignificance of the P-450sinvolved involved in steroid anabolism) or be a catalyst for the oxidation in the processing of normally endogenous chemicals? While it is of many substrates (e.g. some of the inducible P-450s utilized in clear that some of the P-450s such as P-45021A2 are critical for xenobiotic oxidation). There are many different P-450 enzymes; humans, the lack of other P-450s such as P-450 2D6 does not we now realize that there canbe >30 P-450 (or so-called CY,) generally appear tobe a detriment to health(16, 17). Sometimes genes expressing their products in a single organism, and many particular P-450s canbe targets for drugs that function by mechof these are concurrently produced in a single tissue.These genes anism-based inactivation, such as the estrogen-forming aromahave been classified on the basis of their coding sequences (2). tase P-450 19 in breast cancer (18, 19). w-Hydroxylation of fatty acids was the assay that Lu and Coon (20)first used in the Functional Roles of Cytochrome P-450 Enzymes isolation and reconstitution of hepatic microsomal P-450s, but There is broad interest in the P-450s becauseof the significance the physiological contribution of this activity does not appear to of these enzymes in a wide variety of disciplines, ranging from be dramatic in the case of short-chain fatty acids; with longer medical genetics to inorganic chemistry. Historically the P-450 fatty acids and eicosanoids the processes of w - , w-1-, and w-2gene family has been considered to be a very large one, with at hydroxylation may be more important (21). While it is accepted (2). numerous that some steroid hydroxylations are extremely critical (22), the least 38 genes identifiedin the rat to date However, nonspecific hepatic microsomal enzymesthat other cases of large gene families now exist, such as the steroid situation with the S- and UDP-glucuron- also oxidize xenobiotics is unclear; while many can hydroxylate receptors, interferons, and the glutathione osyl transferases involved in drug metabolism (3, 4). There have androgens and other steroidsregio- and stereoselectively (23), it been two major views on the function of P-450 enzymes: (i) the is not obvious that these reactions are particularly critical (24). enzymeshave critical and specific roles in the metabolism of A low K,,, fora particular reaction is not strong evidence of endogeneous chemicals and (ii) the enzymes process the burden inherent selectivity or function, anda low Kd can actually work of natural products (and, in todays world, chemicals such as against catalysis by increasing the activation energy (25). drugs and other xenobiotics, i.e. foreign chemicals) in a relaRecently Spector and others (26) have identifiedmetabolic tively non-selective manner (5).There is certainly ampleevidence pathways in mammals leading to the de novo synthesis of the for the latter view, and a special case can occur when microoranalgesic morphine, and itis highly likely that at least several of ganisms are selected for growth on a particular chemical (or for these steps are catalyzed by P-450s, as might be expected from growth in the presence of a toxic chemical). Thus, Gunsalus and the knownroles of P-450s in oxidizing these (27) and other his associates (6, 7) were able to develop a useful bacterial model nitrogen-containing drugs and alkaloids (28). system with a pseudomonad that harbors a plasmid coding the There is considerable interest in the function of eicosanoids redox system containing P-450 101 (P-450,,,), an enzyme which and other long-chain fatty acid derivatives as messengers and the allows growth of the organism on the terpene camphor as the function of P-450s in relation to the metabolism of these comcarbon source. More recently, Gunsalus and his colleagues (8) pounds. Arachidonic acid is converted to alcohols and to epoxides have also isolated the distantly related P-4501in from bacteria by P-450s (21, 29, 30), and these epoxides can even be incorposelected for growth on another terpene, linalool. As has been rated into phospholipids (31).There is significant evidence that pointed out elsewhere (5, 9), mammals consume a considerable the epoxides of arachidonic acid can exert important physiological daily burden of natural products such as terpenes, steroids, and responses a t low concentrations. Recently Kauser et al. (32) have alkaloids, and P-450 enzymes are involved in much of the clear- shown that P-450 inhibitors can attenuatemyogenic the response ance. In the same way, the P-450s encounter numerousnew drugs of dog renal arteriesin vivo, and sucheicosanoid-linked pathways and pollutantseach year.The lack of complete selectivity of some may be involved. Sucharachidonic acidepoxides have been of the P-450s and the number of the individual forms are thus an implicatedin pregnancy-induced hypertension (33).Although advantage. However, in some cases the oxidationof these chem- some of the less selective hepatic P-450s have been shown to icals can generate dangerous electrophiles that are detrimental account to formuch of the epoxidation of arachidonic acid in microsomal systems (34), the contributionsof individual P-450s * Research in this laboratorywas supported by United States Public Health to in vivo function remain to be defined. Iwai and Inagami (35) Seyice Grants CA 44353, ES 00267, ES 01590, and ES 02205. The abbreviation used is: P-450, cytochrome P-450. havefoundthat geneticallyhypertensive rats fail toexpress

Reactions andSignificance of Cytochrome P-450 Enzymes

10019

10020
F , l

Minireview: P-450 Reactions

RH t i

k.

RH

Fd-0s RH

Feu ROH

(F-OH)~ R.

(51), ring expansion, N-hydroxylation,exchange of protons with solvent (52), and modification of the heme prosthetic group (53, 54) may be understood in these paradigms(39,40, 42).

(FSO~ RH

Other Mechanistic Modes

SCHEME 1. Steps in the oxygen activation and substrate oxidation by P-450 enzymes can catalyzereduction reactionsas well as most P-450s. The Fe in the heme prosthetic group is featured; the substrate (RH) isboundnear the distalligand (site ofoxygen binding). The starting oxidations. Notable examples include carbon tetrachloride (55) binding, reduction,and oxygen oint for the cycle is Fe, followed by substrate Einding. The three intermediatesfol1owingFe-02RHare putative and deduced and azo dyes (56). Other reported reductions (e.g. of epoxides) (57) are poorly understood (1). P-450 enzymes can form H202 primarily from model work and other inferences; the superscript 3+ refers to the overall charge on the entity, the precise localization of which is yet when the oxidationof substrates is not tightlycoupled to electron undetermined. The roleof a transient radical R is best accepted in carbon flow. Evidence has been accumulated that most of this Hz02 is hydroxylation.At the end of the sequence the product ROH is released.

This reaction, analogous to thatof the mitochondrialcytochrome oxidase, appears to be exacerbated in the presence of uncoupling agents such as perfluoroalkanes whose C-F bonds are noteasily broken (58). The i n uiuo significance of these nonproductive reactions is still vague; in liver microsomesand with some purified P-450s a large fraction of the reducing equivalents of NADPH is used nonproductively, and itis not clear that sucha stress on the Mixed Function Oxidation Reaction Chemistry reduced pyridine nucleotide pool is incurred in intact tissues. P-450s can also utilize HZO2,hydroperoxides, and peroxides, Most P-450 reactions proceed with the stoichiometry characand in this regard they have some relationship with the peroxiteristic of monooxygenases (39). dases, which also utilize formal Fe(1V) porphyrin radical cation NAD(P)H + 0, + RH + NAD(P) + Hz0 + ROH intermediates (Fe03) (43, 60). The fungalenzyme chloroperoxiIn many cases the productdoes not appear to be a simple alcohol dase is by spectral definition a P-450; it catalyzes the following because rearrangement has occurred. P-450 has not been shown reaction (61). to act asa lipoxygenase or other type of dioxygenase, e.g. with a HZ02 + HCl + RH + RCI + 2H20 reaction of the type The turnover number for chlorination of dimedone ison theorder 0 of lo5 min-.3 The enzyme can also carry out some of the oxidaRH + R 2RO, + ROOH, tions diagnostic of the more typical P-450s (drug N-demethylaexcept perhaps in thecase of unusually labile compounds. How- tion, oxygenation, etc.) in the presence of Hz02 (62, 63). Oxidaever, P-450s often have the capability to utilize hydroperoxides tions by the hepatic P-450s can also be supported by artificial in various modes (uide infra). oxygen surrogates such as iodosylbenzene (64) The catalytic mechanismof monooxygenation may be considRIO + R + RO + RI eredin two parts, oxygen activationandsubstrateoxidation (Scheme 1). Current views of the details of both aspects have and hydroperoxides. The hydroperoxide-supported reactions are been discussed in detail elsewhere (39-43). The Fe(II)-Oz/sub- complex because P-450s can apparently cleave hydroperoxides strate complex is unstable but hasbeen characterized both with either homolytically or heterolytically, depending upon the parthe bacterialP-450 101 (P-450,,,) and mammalian P-450s (7,44, ticular protein, 45). Some evidence for the other oxygenated complexes has been Fe3++ ROOH + RO- + (FeOH)3f seen with theP-450 enzymes (46, 47), but much of our inference is based upon studies of diagnostic rearrangements catalyzed by Fe3++ ROOH + RO + (FeOH)* the enzymes and biomimetic metalloporphyrin models (39, 48, . . 49). The overalloxygenation reactions include such processes as and in the latter the alkoxide anion may propagate radical hydroxylation at carbon and the heteroatoms N, S, and I, deal- reactions (43, 49). p-450s can also catalyze reductive p-scission kylation Of amines and ethers, and epoxidation (39, 40). These of hydroperoxides to yield alkanes and carbonyl products (65). reactions can all be rationalized in terms of two steps, abstraction The flaxseed p-450 isolated by Song and Brash2 a linolenic Of a hydrogen atom (or electron) and oxygen rebound (radical an allene oxide in what appears to be hydroperoxide to generate recombination). aninternalrearrangement(Scheme 2). Otherinterestingrearrangements of prostaglandin Hz are catalyzed bythe important (FeO)3 + RH + (FeOH)3R + Fe3++ ROH P-450s thromboxane synthase and prostacyclin synthase The chemistry in the different reactions is thought to be rather (Scheme 3). invariant, and the key influence on catalytic specificity is the apoprotein (40). Further, the different P-450 enzymes should not What Regulates Rates o f Catalysis? individually be considered as strictly being epoxidases, N-demeMost of the P-450 reactions are relatively slow, and rates of thylases, etc.; thereaction effected is a function of the fit of the -1 nmol of product formed/nmol of P-450/min (or min) are substrate (or, more properly,its transition state) with the protein. common for many substrates. Rates thisslow can explain in uiuo A single protein can catalyze all of the types of reactions, de- drug disposition in many cases. Rates withsome other substrates pending upon the substrate presented (39). Moreover, seemingly (42,50),ester cleavage unusual reactionssuch as dehydrogenation All rates are expressed as turnover numbers, inunits of min (i.e. nmol of
W-C. Song, and A. R. Brash, submitted for publication.
product formed/nmolof P-450 or metalloporphyrin/min,usually under optimal conditions).

mRNA coding for P-450 4A2; the importance and mechanismof this defect also require further study. Ullrich and his associates (36) have characterized the important enzymes prostacyclin synthase and thromboxane synthase as P-450s which operate via specialized mechanisms involving rearrangement of oxidized chemicals as opposed to oxygen activation per se. These enzymes are critical in influencing many functions. Recently Song and Brash(37, 38) have characterized a flaxseedallene oxide synthase as a P-450 (uide infra). The hydroperoxide substrates are found in high amounts in plants, where the enzyme catalyzes a key step in the biosynthesis of the plant growth hormone jasmonicacid. This activity hasalso been demonstrated in lower animals, including coral and starfish oocytes. These are but a few examples of situations where P-450 enzymes may be considerably important.

formed from the nonenzymatic dismutation of superoxide anion, Or,which is a breakdownproduct of the Fe(II)-O,complex (58). Fe(II)-02+ Fe(II1) + 0;
1/20,+ %H202

However, in some cases superoxide anion has not been detected in the decomposition of P-450 Fe(II)-02 complexes (45). The complete reduction of O2 to HzO by P-450 has also been documented (59). 2NADPH

+ 2H+ +

0 2

+ 2H20

Minireview: P-450 Reactions

10021

AOH

4R '

SCHEME 2. P-450-catalyzed ( A )epoxidation of arachidonate and ( B ) rearrangement of an allylic hydroperoxide to an allene oxide and subsequent products (37,38).

Hd
MI*

o...... I \ . . . . @

"NO
QR2

" 0
f i R

/
/ a

R~~ -aR,
'9

supply can actually limit the rates of P-450 reactions (72). Ultimately all of these features may be understood a t a molecular level. The three-dimensional structures of several kinds of bacterial P-450,., (P-450 101) crystals are known at high resolution (73, 74). However, none of the intrinsic membrane-bound P-450s has been crystallized to date. Extrapolationof the domains of the soluble bacterial protein to the other enzymesmay be useful in illuminating several features of function but will probably not reveal highresolution details of catalytic specificity, which can be highly sensitive to small amino acid replacements (75). The structureof the bacterial protein, however, has revealed a general domain structure and that the iron spin state is controlled by the accessibility of a n H 2 0 residue (or OH-) as a distal iron ligand (76). The same principle probably holds in the other P-450s, but the correlation between spin state, Fe3+/Fe2+ oxidation-reduction potential, and catalysisisclearly not universal among P-450s(77).Negishi and his associates(78)havealso shown that minor variations in a single residue (position 209 of a mouse P-450) can produce dramatic changes in the catalytic specificity toward a pair of substrates, coumarin and testosterone, and in the iron spin state, but there is no obvious correlation. Site-directed mutagenesis experiments have also been used to implicate the basic amino acids (Lys and Arg) clustered in two regions of rat P-450 1A2 (in thevicinity of residues 100 and 450) in electron transfer from NADPH-P-450reductase (79). Ishimura's laboratory(80) has used site-directed mutagenesis to show that (in bacterial P-450 101) the Thr-252 residueis near the distal heme ligand region and is critical for appropriate heterolytic scission of the oxygenated iron complex. Fe2+-02

.d
SCHEME 3.

Proposed mechanism for P-450-catalyzed reand prosarrangements of prostaglandin Hzto thromboxane (TXAz) In the absenceof this residue the enzyme is "uncoupled," that is HHT,12(S)-hydroxy-5,8,10-(Z,E,E)-heptadecatrienoicit acts as an oxidase. tacyclin (PGZ,) (36). acid). NADH H+ 0 2 + NAD+ HZ02

C Y

T X A l

I
C Y S C Y 8

le-

H+

Thr-252 Fe-0-OH

+ (FeO)3+

+ OH-

I
s

such as vitamin D are much slower (13). As mentioned above, This finding is surprising in that the appropriate heterolytic rate of -lo5 min", chloroperoxidase appears to form producta at and the flaxseed P-450 of Song and Brash' converts thelinoleic cleavage was thought to be largely a function of the axial Cys hydroperoxide to alleneoxide at a rate of -80,000 rnin".' Throm- thiolate ligand (73). The question can thenbe raised as to what boxane synthase catalyzes the rearrangement of prostaglandin the function of the universal Cys ligand really is in catalysis. biomimetic models seem to H2 a t -2500 min". Among the P-450 reactions where electron Does it have a function as such? The transfer is actually used in oxygen activation, the catalytically work a t least somewhat effectively without it, although the presfastest enzymes are those in bacteria. P-450 101 (P-450,.,) and ence of imidazole often improves these systems (49). However, the ability of a T h r hydroxyl to assist in such a protonation has P-45Oli. form products at rates >lo3 min" and so does an unusual been questioned by Raag and Poulos, who feel that the role of Bacillus subtilis P-450 which contains its accessory flavoprotein of anetwork of covalently attached to its N terminus (66). Amongthe hepaticP- Thr-252 is better understood in the context 450 enzymes,the use of an oxygen surrogate (e.g. iodosylbenzene) residues near the distalface of the heme (73, 74). instead of NAD(P)H and the reductase can increase rates of N Other Aspects of P-450 and Prospects f o r the Future demethylation by nearly an order of magnitude, to -800 min" (67).Forcomparison,Traylorhas developed a model system In this brief review it is really not possible to deal with all consisting of an iron porphyrin and pentafluoroisodosyl benzene aspects of P-450 biochemistry or to adequately acknowledge all which can carry out oxidations with a turnover numberof 18,000 studies that have led to our currentviews. The reader is referred (73), min" (68). A more complete biomimetic system consisting of a to lead reviews andarticlesonP-450proteinstructure manganeseporphyrin,N-methylimidazole, 02,flavin, and N - nomenclature and sequence similarity (2), biomimetic and other methylnicotinamide (as the electron source) catalyzed the oxi- chemical models (49), catalyticmechanism (39-42), molecular dation of nerol at a rate of 9 min" with a 33% yield of product biology studies (16) and regulation (Bl), features of structure/ based upon reducing equivalents (69). activity relationship (74, 82), and roles of P-450s in steroidogenMuch of the early literatureconsidered the nature of the rate- esis (14), drugmetabolism (83, 84), andcarcinogenesis (10). limiting step in P-450 catalysis. What has emerged is the view Great progress has been made in the understanding of these that the rate-limiting step probably varies depending upon the complex enzymes through the efforts of many individuals and the particular P-450enyzme and substrate. In some cases itis appar- availability of new technologies. Some areas in which research of introduction of the first or second electron is needs remain include the following. (i) Analysis of more protein ent that the rate limiting (Scheme l), and cytochrome bs can play an important structures and their interactions with substrates, in three dimenrole in the transfer of the second electron in some cases (70). In sions, is needed. The P-450s are too large for the application of othersituationstheexistence of large intermolecularkinetic global NMR methods, anda need exists for crystallization of the deuterium isotope effects argues that hydrogen atom abstraction membrane proteins. (ii) Further details of catalytic mechanisms is limiting (51). An argument for rate-limiting product release remain to be elucidated. For instance, some points regarding Nhas been reported (71), and actuallysome in cases (e.g. conversion oxidation and epoxidation remain controversial. (iii) The reguof testosterone to estradiol) a single P-450 cancatalyze sequential lation of levels of P-450 enzymes is complex, and much remains reactions on a substrate (19); the extent of equilibration of the to be learned. The individual P-450s appear to be regulated in product with the medium is unknown. In the liver, the oxygen different manners, with some elements of similarity among cer-

10022

Minireview: P-450 Reactions

tain genes. While much attention has beenfocused on transcription, there are other points of control as well. (iv) There are still important reactions for which the P-450s remain to be isolated and characterized. (v) Finally, in vivo and i n vitro systems are needed to determine the significance of the individual P-450s in influencing humans torisk and disease from xenobioticsand also endogenous chemicals.

wn5-1m3
Y"

" . _ I

Acknowledgments-I thank Drs. J. H. Capdevila, L. J. Marnett, M. J. Coon, 40. T. D. Porter, T. Shimada, andA. Brash for their comments on the manuscript. 41. The reader is referredto a coming minireview on P-450 by Drs. M. J. Coon and T. D. Porter in this journal and to an issueof FASEB J. on P-450s to appear 42. in late 1991. 43.

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