Chiral orientation of prosthetic heme in the cytochrome P-450 active site.

The absolute orientation of the prosthetic heme group in the active site of a hemoprotein may influence its substrate selectivity and catalytic properties. The only method available until now to determine the chiral orientation of the heme in a hemoprotein has been high resolution x-ray crystallography. The orientation of the heme in cytochrome P-450, therefore, is unknown because a crystallographic structure is not available for any form of this enzyme. We report here that the absolute configurations of the N-ethylprotoporphyrin IX adducts formed from the prosthetic hemes of cytochrome P-450 and hemoglobin during catalytic turnover of appropriate substrates are identical. The prosthetic heme in the inactivated cytochrome P-450 enzyme, therefore, has exactly the same orientation, relative to the fifth iron ligand, as the heme in hemoglobin. The approach described here can be used to determine the prosthetic heme orientation in other hemoproteins, including other cytochrome P-450 isozymes, for which x-ray structures are not available.

Major advances have been made in our understanding of the function, structure, and mechanism of cytochrome P-450 enzymes during recent years. The existence of multiple isozymes that differ in molecular weight, chromatographic and spectroscopic properties, tryptic peptide maps, terminal amino acid sequences, immunological reactivity, and substrate specificity is now well established (for reviews see Refs. [1][2][3][4][5]. The complete amino acid sequences for two of the isozymes, one induced in rats by phenobarbital and the other isolated from camphor-grown Pseudomonas putida (P-450,,,), have recently been deciphered (6,7). The structure of the active site itself remains ill defined, however, although it has been established that the active site prosthetic heme group is coordinated through its iron atom to a thiol anion of the protein matrix (8)(9)(10)(11). It is also known that in the absence of substrates a hydroxyl (9,12) or an imidazole (13) occupies the sixth iron coordination site. The catalytic activation of molecular oxygen and its subsequent transfer to substrates occur on the iron atom at the site occupied by the exchangeable sixth ligand.
The asymmetric substitution pattern of protoporphyrin IX * This work was supported by National Institutes of Health Grants GM 25515 and AM 30297. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Research Fellow of the Alfred P. Sloan Foundation (1978)(1979)(1980)(1981)(1982).
To whom correspondence should be addressed. makes the two faces of heme stereochemically distinct (prochiral). Coordination of an achiral ligand to heme gives rise to two enantiomeric complexes or, if the ligand itself is chiral (as in a protein active site), to diastereomeric complexes (Fig. 1). The structure and catalytic specificity of otherwise identical active sites may differ if they incorporate the prosthetic heme in the opposite orientations because: ( a ) the active site structure may be perturbed by the different locations of the vinyl and methyl substituents and ( b ) the electronic structure of the heme may be altered if the angle between the vinyl groups and the a-system of the porphyrin ring differs in the two orientations (14). The absolute orientation of the prosthetic heme moiety is not known for any cytochrome P-450 isozyme because no method other than high resolution x-ray crystallography is now available for determination of the heme stereochemistry. The orientation of the heme is known, of course, in proteins like myoglobin and hemoglobin for which crystallographic structures are available (15,16).
We have demonstrated that cytochrome P-450 is inactivated during catalytic turnover of a variety of substrates, including olefins, acetylenes, and 4-alkyl-1,4-dihydropyridines (17)(18)(19). Enzyme inactivation results from alkylation of the prosthetic heme group by a catalytically activated form of the substrate. We have isolated and unambiguously identified the heme adducts (after removal of the iron atom) as N-alkylprotoporphyrin IX derivatives (19)(20)(21)(22). The N-alkyl group when DDEP' is used as the destructive agent is the ethyl moiety derived from the 4-position of the substrate (19). Preferential alkylation of one of the two faces of prosthetic heme is implied by our observation that the N-alkyl porphyrins obtained with propyne (21) and with the 4-methyl analogue of DDEP (3,sdicarbethoxy-1,4-dihydrocollidine) (23) are optically active, We have been unable, however, to determine the crucial degree of stereospecificity or the absolute orientation of the heme. Our recent discovery that hemoglobin reacts with ethylhydrazine to give the N-ethyl porphyrin also obtained with DDEP (24) provides the key to this stereochemical impasse because the absolute stereochemistry of the hemoglobin adduct can be deduced from the known active site geometry of this hemoprotein. We report here the use of this novel approach to establish the absolute orientation of the prosthetic heme group in the rat cytochrome P-450 isozyme inactivated by DDEP. The chemical reactivity of hydrazine derivatives with heme (25), furthermore, suggests that heme adducts can be used to determine the prosthetic group stereochemistry in other hemoproteins for which crystallographic structures are not available.

EXPERIMENTAL PROCEDURES
N-Ethylprotoporphyrin IX Regioisomers-The four N-ethylprotoporphyrin IX regioisomers formed in phenobarbital-pretreated rats injected with DDEP were isolated as the dimethyl esters and were purified as described previously (19). The procedure for isolation of the same N-ethyl isomers from incubations of hemoglobin with ethylhydrazine has also been reported (24). The samples used in the present study were obtained by stirring hemoglobin ( the corresponding chlorozinc complexes for spectroscopic studies (18,19).
Circular Dichroism Spectroscopy-The individual chlorozinc Nethylprotoporphyrin IX isomers from cytochrome P-450 and from hemoglobin were dissolved in spectroscopic-grade chloroform. The concentration of each solution was adjusted to approximately the same value using a molar absorbance coefficient at 431 nm of 124,000 M" cm" (23). The circular dichroism spectra were recorded on a Jasco J-500A spectropolarimeter at 23 "C (2-cm cells, time constant 4, 2.5 cm/min). The spectra were corrected by subtraction of the baseline observed in the absence of the porphyrins.

RESULTS AND DISCUSSION
The four regioisomers of N-ethylprotoporphyrin IX are isolated from phenobarbital-pretreated rats injected with DDEP but one of these, the isomer with the N-alkyl group on pyrrole ring A, is quantitatively the most important (19). The same four regioisomers are obtained in the reaction of ethylhydrazine with human hemoglobin except that here there is a strong preference for N-alkylation of the nitrogen in pyrrole ring C. The spectroscopic comparison of the porphyrins obtained from cytochrome P-450 and hemoglobin has been made with one single pure regioisomer because the light rotatory properties of the N-ethylprotoporphyrin IX chromophore depend on the nitrogen which bears the N-alkyl group (see discussion below). The isomer alkylated on pyrrole ring C, shown by NMR to be free of contamination by other isomers (19), has been chosen for this purpose. The circular dichroism spectra of the ring C isomers derived from the two hemoproteins are identical in sign and amplitude (Fig. 2). The two Nethyl porphyrins, therefore, not only have the same absolute configuration but also, in view of the identical molar ellipticity values, are of equal optical purity.
The absolute configuration of the heme prosthetic group in hemoglobin is that on the left in Fig. 1 (15). We have recently established that carbon radicals are liberated in the reaction of alkyl and aryl hydrazines with heme-bound oxygen and have shown that, at least in the case of phenylhydrazine, the phenyl group is covalently bound to the heme iron before it undergoes a 1,2-shift to one of the porphyrin nitrogen atoms (24,26). The N-alkyl function in hemoglobin consequently is introduced from the side of the porphyrin to which molecular oxygen is bound. The ring C adduct from hemoglobin, therefore, can be assigned the R absolute configuration shown in Fig. 3. Alkylation of the cytochrome P-450 prosthetic heme during suicidal metabolism of DDEP and other substrates also requires that alkylation occur from the side of the heme which faces the activated oxygen species. The heme prosthetic group of the major isozyme induced by phenobarbital (an isozyme highly vulnerable to destruction) (19) consequently must, like hemoglobin, be oriented exclusively as shown on the left in Fig. 1. If the prosthetic group were present in two different orientations, the circular dichroism bands of the adduct should have been smaller in amplitude than those of the hemoglobin-derived porphyrin due to the inverse absorp- tion of the opposite enantiomer.' For the same reason, the prosthetic heme must also have the same absolute orientation in all the other minor but still quantitatively significant hepatic isozymes destroyed by DDEP in phenobarbital-pretreated rats.
The importance of using a single pure regioisomer of the heme adduct in studies of this kind is emphasized by our finding that the circular dichroism spectra of the four isomers of N-ethylprotoporphyrin IX obtained from cytochrome P-450 are different (not shown). The sign of the strong circular dichroism peak at approximately 435-445 nm, for example, is positive for the zinc complexes of the isomers alkylated on rings A, C, and D (isomers 11,111, and IV, respectively, in Ref. 19) but is negative for the isomer alkylated on ring B (isomer I). The signs and positions of other peaks in the spectra of the isomers also differ. The circular dichroism spectrum reported previously for the ring A isomer of N-(2-oxopropyl)-' The two enantiomers of N-ethylprotoporphyrin IX, like all enantiomers, are expected to have circular dichroism spectra that are identical but of opposite sign.

I
Chuen-Shang C. Wu for access to the Jasco spectropolarimeter and assistance in its use. protoporphyrin IX (21) obtained with propyne, however, is very similar to that of the ring A isomer of the N-ethyl structure obtained here. It can now be assigned the absolute stereochemistry expected if' alkylation occurs from the same side as the addition of the N-ethyl in Fig. 3. The circular dichroism spectrum of the ring D3 isomer of N-(2-hydroxy-ethy1)protoporphyrin IX obtained with ethylene (20) is likewise virtually the same as that of the corresponding N-ethyl derivative (not shown) and must also have arisen by addition of the substrate to the heme from the same side.
The method developed for the present study is applicable not only to the study of cytochrome P-450 enzymes but also, if appropriate suicide substrates are used, to other hemoproteins that have a vacant coordination site (or an exchangeable ligand) on the prosthetic heme iron. The fact that N-alkylation occurs during the reaction of hydrazine derivatives with hemin itself (25) suggests that compounds of this class may be used to explore the orientation of heme in other hemoproteins, including the peroxidases and various carrier proteins.