Cytochrome 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. 

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.

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). 

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). 

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. 

The cytochrome P-450 enzyme system that has been characterized structurally is the form cytochrome P-450cam, which catalyzes the oxidation of the monoter-... 

Many of the P-450 enzymes have been difficult to characterize, because they are membrane-bound and consequently relatively insoluble in aqueous solution. However, cytochrome P-450cam> which is a component of the camphor... 

In the enzymatic reactions themselves, there is also strong evidence to support a stepwise mechanism involving free-radical intermediates. For example, cytochrome P-450cam gives hydroxylation of c -camphor only in the 5-exo position, but deuterium-labeling studies show that either the 5-exo or the 5-endo hydrogen is lost (Reaction 5.78). ... 

Davydov, R., T.M. Markis, V. Kofinan, D.E. Werst, S.G. Sligar, and B.M. Hoffinan (2001). Hydroxylation of camphor by reduced oxy-cytochrome P-450cam Mechanistic implications of Epr and Endor studies of catalytic intermediates in native and mutant enzymes. J. Am. Chem. Soc., 123, 1413-1415. 

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