of the Carbon Monoxide Complex of Ferrous Chloroperoxidase”

SUMMARY The absorption spectrum of the carbon monoxide complex of ferrous chloroperoxidase from Caldariomyces fumago has been shown to be quite similar to the characteristic spectrum of CO complexes of cytochromes of the P-450 type. Comparison of other spectral properties of chloroperoxidase and cytochrome P-450,, reveals a striking resemblance between the two proteins. The Soret absorption maxima for native, reduced, cyanide, nitrous oxide, and N-phenylimidazole complexes are quite similar. N-Phenylimidazole, a potent inhibitor of the cytochrome P-450,,-catalyzed hydroxylation of camphor, is a very effective inhibitor of a chloroper-oxidase-catalyzed peroxidation reaction. Like P-450, chloroperoxidase undergoes characteristic spectral changes in the presence of substrates and nitrogenous compounds. Type I and type II spectral changes have been observed. Care-fully controlled denaturation of chloroperoxidase resulted in the formation of a species having a spectrum essentially identical with that of cytochrome P-420, the denatured form of P-450. The spectral similarities described here indicate that both proteins quite for the heme prosthetic group. Both proteins with physical


Illinois 61801 SUMMARY
The absorption spectrum of the carbon monoxide complex of ferrous chloroperoxidase from Caldariomyces fumago has been shown to be quite similar to the characteristic spectrum of CO complexes of cytochromes of the P-450 type. Comparison of other spectral properties of chloroperoxidase and cytochrome P-450,, reveals a striking resemblance between the two proteins.
The Soret absorption maxima for native, reduced, cyanide, nitrous oxide, and N-phenylimidazole complexes are quite similar.
N-Phenylimidazole, a potent inhibitor of the cytochrome P-450,,-catalyzed hydroxylation of camphor, is a very effective inhibitor of a chloroperoxidase-catalyzed peroxidation reaction. Like P-450, chloroperoxidase undergoes characteristic spectral changes in the presence of substrates and nitrogenous compounds. Type I and type II spectral changes have been observed.
Carefully controlled denaturation of chloroperoxidase resulted in the formation of a species having a spectrum essentially identical with that of cytochrome P-420, the denatured form of P-450.
The spectral similarities described here indicate that both proteins provide quite similar environments for the heme prosthetic group. Both proteins also compare favorably with respect to physical properties such as molecular weight, high content of acidic amino acids, and low isoelectric point.

Cytochrome
P-450 is an unusual CO-binding hemoprotein which was first observed in rat liver microsomes (l-3).
It has been shown to function as the terminal oxidase (4,5) in a mixed function oxidase system which is involved in the metabolism of fatty acids, steroids, drugs, carcinogens, and other foreign compounds (6,7). An unusual characteristic of cytochromes of the P-450 type is the position of the Soret band of the reduced CO complex at an extremely long wave length.
The Soret peak for CO complexes of ferrous P-450 hemoprotein occurs in the 450-nm range while the position of the Soret peak for the reduced CO complexes of most hemeproteins is approximately 420 nm (1). Proteins displaying this P-450 anomalous behavior have since been shown to be present in various mammalian tissues (5), as well as in yeasts (8), plants (9), and bacteria (10).
The extreme catalytic diversity and lack of substrate specificity exhibited by these enzymes is evidenced by their ability to catalyze the hydroxylation of aromatic compounds and alkanes, the dealkylation of secondary and tertiary amines, and the osidation of primary amines (11). These reactions are thought to involve insertion of a hydroxyl group into the substrate and may result in the demethylation of substrates such as nitroanisole, aminopyrine, and N-methyl aniline (12). Cytochrome P-450 from several sources is currently under study in numerous laboratories.
The mechanism of the hydroxylation reaction catalyzed by this class of enzymes and the manner in which these enzymes activate oxygen are under intense scrutiny.
Most of these studies have been handicapped by the inability to isolate readily soluble protein without resorting to detergent solubilization (13), and by the inherent instability of the enzyme. Cytochrome P-450 readily denatures to a catalytically inactive P-420 form (3,(10)(11)(12)(13).
Cytochrome P-450,,, from Pseudomonas putida, which has been isolated by Gunsalus and co-workers (14) and also has been studied by Peterson (15), is the only cytochrome P-450 which can be isolated in a soluble form without resorting to detergents or other solubilizing agents. We report here the striking similarities between chloroperoxidase, a halogenating hemeprotein which has been extensively studied in our laboratory, and cytochrome P-450,,,.

EXPERIMENTAL PROCEDURE
The isolation of chloroperoxidase from Caldariomyces junlago has been reported previously (16). Preparations used for the experiments reported in this paper had specific activities greater than 2000 units per mg and R, values greater than 1.40, indicating that these preparations were at least 95% pure. The visible spectra were recorded on a Cary 15 spectrophotometer using cells with path lengths of 1 cm. Thunberg cuvettes which had been degassed and flushed with nitrogen four times before the anaerobic addition of an excess of sodium dithionite were used for the reduced spectral recordings of chloroperoxidase.
For the reduced CO-enzyme complex, the Thunberg cuvette was flushed with CO either before or after the addition of dithionite.
The sequence of CO addition had no effect on the final spectrum.
The assay for the oxidation of thiourea has been described previously (17). N-Phenylimidazole was a gift from Dr. I. C. Gunsalus and .John Lipscomb.
Sodium dit'hionite was obtained from the J. T. Bakei Co., CO from Union Carbide, and all other chemicals were reagent grade available from commercial sources.

AP\'D DISCUSSION
Recently, we began an intensive study of the optical, ESR, and Mossbauer properties of chloroperoxidase and its complexes in order to learn more about the environment of the heme and the changes it undergoes upon binding substrates and during catalysis.
As shown in Fig. 1, chloroperoxidase readily forms a reduced enzyme-CO complex when the enzyme is reduced by a 2-fold molar excess of sodium dithionite under a carbon monoxide atmosphere.
Formation of the reduced CO-chloroperoxidase complex is a fully reversible process, when air is admitted to the cuvette the nat'ive oxidized enzyme spectrum is rapidly reformed. The ferrous enzyme-CO complex exhibits a Soret peak at an exceptionally long wave length (approximately 443 nm). This absorption at abnormally long wave lengths is characteristic of  I  I  I  I  I  I  I  I  I . The spectrum of native chloroperoxidase exhibits a Soret maximum at 396 nm with additional peaks at 515 and 650 nm and strongly resembles that of the enzyme-substrate complex of cytochrome P-450,,,. P-450,,,, in the presence of camphor, has a Soret maximum at 391 nm, a peak at 500 with a shoulder at 540 and a peak at 645 nm (11).
Further spectral similarities between chloroperoxidase and P-450,,, become apparent upon comparison of the spectra of various complexes of the two proteins.
As can be seen in Table  I, the Soret positions and extinction coefficients of the two proteins display a good correspondence for both oxidation states and for complexes with ligands (18), such as CN-, NO, and N-phenylimidazole.
In addition to forming characteristic spectral complexes with P-450,,, and chloroperoxidase, N-phenylimidazole is an extremely effective inhibitor of P-450-catalyzed camphor hydroxylation and of chloroperoxidase-catalyzed peroxidations. The inhibition of P-450,, by N-phenylimidazole is competitive with camphor and has a Ki of approximately 1 X 10m7 M.l As seen in Fig. 2, N-phenylimidazole is also a potent inhibitor of the chloroperoxidase-catalyzed oxidation of thiourea. When Nphenylimidazole is added to the assay mixture, there is an initial lag period during which there is no oxidation of thiourea.
After the lag period the oxidation of thiourea proceeds at a rate which is essentially the same as that in the absence of inhibitor.
The results depicted in Fig. 2 indicate that the length of the lag period is roughly proportional to the amount of N-phenylimidazole which was added initially to the reaction mixture.
These results -T suggest that N-phenylimidazola may serve as an oxidizable substrate and that the release of inhibition may be due to osidation of N-phenylimidazole to a compound which cannot exhibit an inhibitory effect on thiourea oxidation.
The addition of a wide variety of substrates and inhibitors to cytochrome P-450 causes characteristic changes in the optical absorption spectrum which reflect changes in the environment and electron density of the heme (19). The types of changes elicited fall into two basic categories (19) which are generally referred to as type I and type II spectral changes. Type I changes involve movement of the Soret peak to shorter wave lengths and are usually detected by the appearance of a peak at 385 to 395 nm in the difference spectrum.
Type II changes correspond to a shift of the Soret to longer wave lengt,hs and the formation of a peak at 420 to 430 nm in the difference spectrum. As shown previously, chloroperoxidase undergoes distinct spectral changes upon the formation of chloroperoxidase-halide complexes (20). The chloroperoxidase-iodide complex shows a typical P-450 type I difference spectrum while the chloroperoxidasechloride complex has a P-450 type II difference spectrum (20). Table II lists the spectral type of chloroperoxidase complex elicited with several ligands.
As with cytochrome P-450, the addition of aniline or pyridine to chloroperoxidase results in typical type II spectral changes (Table II).
Early attempts to solubilize liver microsomal cytochrome 1 I. C. Gunsalus, Personal communication.
P-450 resulted in the conversion of the enzyme to a denatured, catalytically inactive form exhibiting a reduced CO spectrum typical of the b-type cytochromes which have a Soret peak at 420 instead of 450 nm. This denatured form was called P-420 (3). Most P-450 preparations contain some P-420 and, in general, cytochrome P-450 can be converted to P-420 by treatment with denaturing agents such as detergents, proteases, or alcohols (3). As shown in Fig. 1, the reduced CO complex of chloroperoxidase can be converted by alkali denaturation to a species having a Soret peak at 420 nm. The enzyme was denatured by adding small aliquots of sodium hydroxide to the reduced chloroperoxidase-CO complex to raise the pH of the solution from pH 7 to 7.15. Visible absorption spectra (380 to 500 nm) have been taken at different time intervals to monitor the progress of the P-450 to P-420 conversion.
The presence of clearly defined isosbestic points suggests that no intermediates are involved in the denaturation process. The rate of denaturation increases with increasing pH. Conversion of P-450 to P-420 can be stopped at any point by lowering the pH. However, all attempts to convert P-420 back to P-450 by adjusting the complex to lower pH values or by the addition of mercaptides to the complex have been unsuccessful. The over-all absorption spectrum of the chloroperoxidase P-420 species is quite similar to that formed from cytochrome 1'.450,,.
The 01-and P-bands for chloroperoxidase P-420 are at 569 and 539 nm, which compares favorably with the positions for P-420,,, of 565 and 538 nm (3).
A comparison of the physical properties of the two proteins gives further support to the similar nature of these two enzymes. Both are monomeric proteins having similar molecular weights (chloroperoxidase approximately 40,000; P-450,,, approximat,ely 45,000), exhibit a predominance of acidic amino acids over basic residues, and have fairly low isoelectric points (approximately 4 for chloroperoxidase and 4.55 for P-450,,,) (16,21). Unlike most heme proteins, P-450,,, and chloroperoxidase display unusually large rhombic distortions in their high spin forms. ESR studies show the g values for P-450,,, to be 7.81, 3.93, and 1.77 (22) which are quite similar to the g values of 7.44 and 4.302 which have been recently observed for chloroperoxidase.
The striking similarities described here suggest that chloroperoxidase and P-450,, provide quite similar environments for the heme prosthetic group, and imply that both the proteins may have the same axial ligands on the heme iron and almost identical heme-protein interactions.
The atypical character of the spectrum of the reduced CO complexes of these two proteins and other 2 The presence of manganese i n the sample makes EPR analysis difficult i n the vicinity of g = 2.
The EPR studies were done i n collaboration with 5. Peisach and W. E. Blumberg. P-450 cytochromes suggest that these proteins differ from other heme proteins iu having unique structures for one or both axial ligands or alternatively perhaps the P-450 types differ from other heme proteins in more subtle heme-protein interactions.
Whatever the source of this abnormal behavior, it is extremely interesting and is currently under study from a variety of points of view.
In addition to structural similarities as evidenced by spectral analogies, peroxidases and cytochrome P-450.type enzymes share common functional relations.
Peroxidases are able to catalyze a number of oxidations using molecular oxygen as the electron acceptor (23). Since several of these reactions are inhibited by CO, and a ferroperoxidase-CO complex has been observed during the reaction (24), the over-all reaction is thought to involve a ferric-ferrous valency change similar to that involved in P-450 hydroxylations (25). Further support for a structural and functional relationship between cytochrome P-450 and peroxidases comes from the work of Hrycay and 0'13rien (26, 27) who have reported that cytochrome P-450 is responsible for most of t,he peroxidase activity in liver microsomes.
The results of oxygen binding studies with horseradish peroxidase led Wittenberg et al. (28) to propose that horseradish peroxidase might serve as a prototype for a terminal oxidase. However, the reduced CO complex of horseradish peroxidase, as well as lactoperoxidase and catalase, displays the Soret band at the usual 420 nm. 3 One reaction mechanism suggested for cytochrome P-450catalyzed hydroxylation involves the generation of an enzymebound OH+ cation from a hydroperoxide intermediate which could then serve as the active hydroxylating species (29). Studies in our laboratory with IsO-labeled peroxy acids, hydrogen peroxide, and water have led us to postulate that chloroperoxidase-compound I contains a single substrate oxygen atom which can behave like an OHf species (30).
The results presented here suggest that chloroperoxidase may be a valuable adjunct to aid in the understanding of the functional importance of those structural properties which are unique to cytochrome P-450.
These studies therefore should lead to a better understanding of the mechanisms of oxygen activation and hydroxylation.
The functional similarities and possible structural correlates between chloroperoxidase and P-450 are currently under study in our laboratory. This work will be facilitated by the ready availability of gram quantities of crystalline chloroperoxidase.