P-450cam

Atkins, W.M. and Sligar, S.G. (1988) Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate. Biochemistry, 27 (5), 1610-1616. 

Dihaloelimination has also been observed under partially aerobic conditions [274]. With cytochrome P-450CAM as a primary catalyst, dichloroelimination from hexa-, penta-, and 1,1,1,2-tetrachloroethane were catalyzed, and the products were PCE, TCE, and 1,1-DCE, respectively no reaction was observed with TeCA. Significant rates were observed for these reactions at 5% oxygen concentration. 

Poulos, T. L. Howard, A. J. (1987). Crystal structures of metyrapone-and phenylimidazole-inhibited complexes of cytochrome P-450cam. Biochemistry, 26, 8165-74. 

Poulos, T.L., Finzel, B.C. Howard, A.J. (1987). High resolution crystal structure of cytochrome P-450cam. Journal of Molecular Biology, 195, 687-700. 

Raag, R. Poulos, T. L. (1991). Crystal structures of cytochrome P-450cam complexed with camphane, thiocamphor, and adamantane factors controlling P-450 substrate hydroxylation. Biochemistry, 30, 2674-84. 

Schlichting I, Jung C, Schulze H. Crystal structure of cytochrome P-450cam complexed with the (lS)-camphor enantiomer. FEBS Lett 1997 415 253-257. 

Jones JP, Trager WF, Carlson TJ. The binding and regioselectivity of reaction of (R)-nicotine and (S)-nicotine with cytochrome-P-450cam - parallel experimental and theoretical-studies. J Am Chem Soc 1993 115 381-387. 

Fig. 5-6. Crystal structure of the P-450cam reaction site. Reproduced with permission from Loew et at. (1986).

Contzen J, Jung C. Changes in secondary structure and salt finks of cytochrome P-450cam induced by photoreduction A Fourier transform infrared spectroscopy study. Biochemistry 1999 38 16253-60. 

Figure 6.2. Scematic presentation of the Cytochrome P 450cam active site (a) and the Breslow model of steroid hydroxylase (Groves, 1997). Reproduced with permission.

D of protoporphyrin-IX depends on the steric constraints of the substrate binding pocket. Ortiz de Montellano (59) has used this selectivity to probe the active site structure of several heme enzymes. The structure of phenyl-cyt P-450cam has been determined by X-ray crystallography and indicates that N-phenyl heme formation is an accurate, low-resolution probe of active site structure. 

FIGURE 17.24. Active site of cytochrome P-450cam showing Cys-357 and camphor in the active site. The sulfur atom of Cys-357 is indicated by a large stippled circle. 

Crystal structures have been reported for the enzyme with several bound drugs. Metyrapone- and 1-, 2- and 4-phenylimidazole-inhibited complexes of cytochrome P-450cam have each been refined to 2.1 A resolution. Except in the 2-phenylimidazole complex, each complex forms an N Fe interaction to the heme iron atom. In the 2-phenylimidazole complex, water or hydroxide coordinates the heme iron atom and the inhibitor binds in the camphor pocket. Details of the inhibitor binding are shown in Figure 17.25. Eukaryotic cytochrome P-450 is membrane bound and has a different structure from the soluble... 

FIGURE 17.25. Interactions of cytochrome P-450cam with (a) camphor, (b) 1-phenylimidazole. (c) 2-phenylimidazole, and (d) metyrapone. Note that the enzyme-bound Fe does not interact with a ring nitrogen atom in (c) for steric reasons. 

Poulos, T, L. The crystal structure of cytochrome P-450cam In Cytochrome P450 Structure, Mechanism, and Biochemistry. (Ed., Ortiz de Montellano, P. R.) Ch 30, pp. 505-523. Plenum, New York, London (1986). 

Atkins. W M., and Sligar, S G, Tyrosine-96 as a natural spectroscopic probe of the cytochrome P-450cam active site. Biochemistry 29, 1271-1275 (1990). 

Raag, R., and Poulos, T. L. The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450cam- Biochemistry 28, 917-922 (1989). 

Figure 30. Natural catalytic cycle for the hydroxylation of organic compounds catalyzed by the Cytochrome (Cyt) P-450cam monooxygenase system (Cyp = Cyt. P-450 hemeprotein Pdx = putidaredoxin).

The understanding of the degradation of natural products such as camphor has been greatly enhanced by understanding the catalytic cycle of the cytochrome P-450 enzyme P-450cam in structural detail.3,4 These enzymes catalyze the addition of 02 to nonactivated hydrocarbons at room temperatures and pressures - a reaction that requires high temperature to proceed in the absence of a catalyst. O-Methyltransferases are central to the secondary metabolic pathway of phenylpropanoid biosynthesis. The structural basis of the diverse substrate specificities of such enzymes has been studied by solving the crystal structures of chalcone O-methyltransferase and isoflavone O-methyltransferase complexed with the reaction products.5 Structures of these and other enzymes are obviously important for the development of biomimetic and thus environmentally more friendly approaches to natural product synthesis. 

Epoxidation of various olefins by cytochrome P-450 enzymes has been studied using rat liver microsomes [29,30] as well as using enzymes from microbial origin. For example, Ruettinger and Fulco [31] reported the epoxidation of fatty acids such as palmitoleic acid by a cytochrome P-450 from Bacillus megaterium. Their results indicate that both the epoxidation and the hydroxylation processes are catalyzed by the same NADPH-dependent monooxygenase. More recently, other researchers demonstrated that the cytochrome P-450cam from Pseudomonas putida, which is known to hydroxylate camphor at a non-activated carbon atom, is also responsible for stereoselective epoxidation of cis- -methylstyrene [32]. The (lS,2R)-epoxide enantiomer obtained showed an enantiomeric purity (ee) of 78%. This result fits the predictions based on a theoretical approach (Fig. 2). 

During recent years, several cytochrome P-450 enzymes have been cloned and overexpressed in various hosts [33] and studies aimed to modify the active site topology of these enzymes, i.e. of the P-450cam for example, have been... 

Fig. 2. Stereoselective epoxidation of ds-)5-methylstyrene using cytochrome P-450cam from...

The cytochrome P-450cam is able to bring about the stereoselective epoxidation of cis-methylstyrene to the 1S,2R epoxide (Ortiz de Montellano et al. 1991). 

Koga, H., H. Aramaki, E. Yamaguchi, K. Takeuchi, T. Horiuchi, and I.C. Gunsdalus. 1986. camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon. J. Bacteriol. 166 1089-1095. 

P-450cam is able to hydroxylate the -CH3 group of the quaternary methyl group of 5,5-difluorocamphor (Figure 6.14a) to the 9-hydroxymethyl compound (Eble and Dawson 1984), and ada-mantane and adamantan-4-one at the -CH quaternary carbon atom (Figure 6.14b) (White et al. 1984). 

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