Cytochrome camphor hydroxylation

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

Gelb, M. H., Heimbrook, D. C., Malkonen, P., and Sligar, S. G. Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monoxygenase system. Biochemistry 21, 370-377 (1982). 

Zurek, J., Foloppe, N., Harvey, J.N., Mulholland, A.J. Mechanisms of reaction in cytochrome p450 Hydroxylation of camphor in p450CAM. Org. Biomol. Chem. 2006,4(21), 3931-7. 

Alton A, Shaik S, Thiel W (2007) What is the active species of cytochrome p450 during camphor hydroxylation QM/MM studies of different electronic states of compound I and of reduced and oxidized iron-oxo intermediates. J Am Chem Soc 129 8978... 

Heme-coupled monooxygenases contain cytochrome P-450. They are present in microsomes and are responsible for many hydroxylation reactions, e.g. llp-hydroxylation of steroids in the adrenal cortex, 2-hydroxylation of estrc ens in the liver the liver system is especially important in the hydroxylation of drugs and xenobiotics, thus rendering them water soluble, capable of conjugation and easily excreiable. A cytochrome P-450 system responsible for the hydroxylation of camphor (a 5-exo-hydroxylase) has been purified from Pseudomonas putida, and named putidaredoxin it contains FAD, an Fc2S2CyS4 center, and a P-4S0 cytochrome substrate hydroxylation is coupled to the oxidation of NADPH. 

Some hydroxylation reactions catalyzed by cytochromes P450. Hydroxylation of camphor 1 by P450 g from P. putida [6] Hydroxylation of (S)-/V-methylcoclaurine 3 by CYP80B1 in morphine biosynthesis [7] Hydroxylation of 6-deoxyerythronolide B 5 by P450j from Saccharopolyspora erythraea [8] Hydroxylation of cinnamic acid 7 by CYP73 from Arabidopsis thaliana [9] Hydroxylation of diclofenac 9 by human CYP3A4 [10],... 

Scheme 10.2 Cytochrome P450cam-catalyzed hydroxylation of camphor.

Eble KS, JH Dawson (1984) Novel reactivity of cytochrome P-450-CAM methyl hydroxylation of 5,5-difluoro-camphor. J Biol Chem 259 14389-14393. 

Davydov, R., Makris, T.M., Kofman, V., Werst, D.E., Sligar, S.G. and Hoffman, B. M. (2001) Hydroxylation of camphor by reduced oxy-cytochrome P450cam mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes. Journal of the American Chemical Society, 123, 1403-1415. 

This agrees very much with the model proposed by Peterson et al. (8) for the 5-exo hydroxylation of d and 1 camphor in mammalian cytochrome P-450/ and is also consistent with the relationship they noted from steroid metabolism by cytochrome P-450/ between the position hydroxylated and its relation to a polar funtional group. 

Fig. 8. Hydroxylation of camphor deuterated at C(5), and substrate binding in the active site of cytochrome P450cam...

The porphyrin-iron(III)-peroxo complex [Fe(TPP)02] (163) was prepared by the reaction of K02 with Fen(TPP) in the presence of a crown ether, and characterized by spectroscopic methods [p(0—O) = 806 cm-1]542. This peroxo complex (163) was found to be inactive toward hydrocarbons. However, addition of excess acetic anhydride to (163) dissolved in a benzene-cyclohexane mixture results in the formation of cyclohexanol and cyclohexanone. This reaction is thought to proceed via acylation of the peroxo group, giving iron percarboxylate (164), which decomposes to an Fev-oxo compound (165) capable of hydroxylating alkanes.543 Such a mechanism has been suggested for the hydroxylation of camphor by Pseudomonas cytochrome P-450.544... 

Harris D, Loew G. Prediction of regiospecific hydroxylation of camphor analogs by cytochrome-P450(Cam). J Am Chem Soc 1995 117 2738-2746. 

Cytochrome P-450 found in bacteria and lever microsomes is a kind of hemoprotein that activates molecular oxygen to catalyze the hydroxylation of organic compounds in drug metabolism. For example, camphor is incorporated into cytochrome P-450 (forming P-450 cam) and then oxygenated to form 5-exo-hydroxycamphor as shown in Reaction 8 (25). The substrate specificity of this enzyme is not strict. How-... 

Schoneboom JC, S Cohen, H Lin, S Shaik, W Thiel (2004) Quantum mechanical/molecular mechanical investigation of the mechanism of C-H hydroxylation of camphor by cytochrome P450(cam) Theory supports a two-state rebound mechanism. J. Am. Chem. Soc. 126 (12) 4017-4034... 

The cytochrome / 450 responsible for the l droxylation of camphor by Pseudomonas putida has been isolated, and is. in contrast to most others, stable. However this enzyme will only hydroxylate substrates closely related to camphor, and the site of hydroxylation may vary with substrate. Thus camphor gives the exo-S-hydroxy derivative but 5,5-difluorocamphor gives the 9-hydroxy derivative. ... 

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