Camphor cytochrome

Paulsen, M.D. Orn stein, R.L. A 175-psec molecular dynamics simulation of camphor-bound cytochrome p-45Proteins 11 184-204, 1991. 

Scheme 10.2 Cytochrome P450cam-catalyzed hydroxylation of camphor.

They are widely distributed across species. Bacteria possess cytochrome P450s, and P450cani (involved in the metabolism of camphor) of Pseudomonas putida is the only P450 isoform whose crystal stmcture has been established. 

The oxidation of f-butyl methyl ether to f-butanol (Steffan et al. 1997), which is also mediated by the cytochrome P450 from camphor-grown Pseudomonas putida CAM, but not by that from Rhodococcus rhodochrous strain 116. 

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. 

There are two types of electron transport those involving flavoproteins and iron-sulfur proteins, and those requiring only flavoproteins. The X-ray crystal structure of the soluble cytochrome P450 from Pseudomonas putida grown on camphor (P-450-CAM) has been determined (Poulos et ah, 1985), as have several others. The haem group is deeply embedded in the hydrophobic interior of the protein, and the identity of the proximal haem iron ligand, based on earlier spectroscopic studies (Mason et ah, 1965) is confirmed as a specific cysteine residue. 

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. 

Figure 16 X-Ray structure of the active site of cytochrome P450cam. In the camphor-bound form the bond distances are Fe-S 2.20 A Fe-N 2.05 A.33 The heme geometry of the camphor-free form is substantially similar to that of the camphor-bound form the only difference is that the iron atom is displaced from the tetrapyrrole plane towards the axial sulfur atom by 0.30 A in the camphor-free form, and by 0.44 A in the camphor-bound form...

Binding of the substrate camphor (RH) by the free ferric cytochrome (Fe " ) ... 

Fig. 6. Effect of the concentration of camphor and the ionic strength on the fraction of bacterial cytochrome / -450 in the high-spin state (Xhs) in aqueous solution at pH 7 in 25 mAf sodium cacodylate buffer at 18°C. , No added KCl 9. 50 mAf KCI.

Figure 7.13 Cytochrome P450 catalytic site with camphor—the dioxygen intermediate (PDB 1DZ8). Visnalized using CambridgeSoft Chem3D Ultra 10.0 with notations in ChemDraw Ultra 10.0. (Printed with permission of CambridgeSoft Corporation.)...

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. 

This enzyme [EC 1.14.15.1], also known as camphor 5-exo-methylene hydroxylase, and cytochrome P450-cam, catalyzes the reaction of (+)-camphor with putidare-doxin and dioxygen to generate (-F)-exo-5-hydroxy-camphor, oxidized putidaredoxin, and water. A heme-thiolate acts as a cofactor. The enzyme can also utilize ( )-camphor as a substrate, and l,2A-campholide will result in the formation of 5-exo-hydroxy-l,2-campholide. V. Ullrich W. Duppel (1975) The Enzymes, 3rd ed., 12, 253. 

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). 

It has been clearly established that Pseudomonas putida which has been selected to grow on camphor possesses an oxidative metabolic system (54). It contains a hemoprotein, cytochrome Qrn>... 

Figure 18-23 (A) Stereoscopic oc-carbon backbone model of cytochrome P450cam showing the locations of the heme and of the bound camphor molecule. (B) View in the immediate vicinity of the thiolate ligand from Cys 357. From Poulos et al.i9S...

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... 

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