Cytochrome P-450-CAM

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

RS-) of a cysteinyl residue. In fact, the v(Fe—S-) vibration of cytochrome P-450 cam (camphor as the substrate) (B-state) has been observed at 351 cm-1 by Champion et al. (10). The oxidation state marker band of C-state was observed at 1,346 cm-1 by Ozaki et al. (11). It is much lower than the corresponding band of deoxy-Hb at 1,356 cm. This marked lowering has been attributed to the strong jr-basicity of the thiolate ligand, which donates electrons via the Fe (Jjr)-porphyrin (pn ) overlap. As stated in the preceding section, the v(C>2) of oxy-Hb has not been observed by Raman spectroscopy. However, Bangcharoenpaurpong et al. (12) were able to observe the v(C>2) of cytochrome P-450 cam (D-state) at 1,140 cm-1 in RR spectra (420 nm excitation). 

Figure 1. MCD (top) and UV-visible absorption (bottom) spectra at 4 °C of the bis (hydroxymethyl) methylphosphine complexes of ferric chloroperoxidase (CPO) (-----) and cytochrome P-450-CAM (------). MCD spectra...

A recombinant strain of Pseudomonas sp. containing the camphor plasmid (CAM) and toluene dioxygenase genes reduced 1,1,1-tet-rachloro-2,2-difluoroethane to l,l-dichloro-2,2-difluoroethene using cytochrome P-450 cam toluene dioxygenase may then oxidize this to oxalate (Hur et al. 1994 Wackett et al. 1994). 

Sono, M. and J.H. Dawson (1982). Formation of low spin complexes of ferric cytochrome P-450-CAM with anionic ligands Spin state and ligand affinity comparison to myoglobin. J. Biol. Chem. 257, 5496-5502. 

Glascock MC, Ballou DP, Dawson JH (2005) Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-ini-tiated turnover of cytochrome P-450-CAM probing the effector role of putidaredoxin in catalysis. J Biol Chem 280 42134 2141... 

Sono M, Andersson LA, Dawson JH (1982) Sulfur donor ligand binding to ferric cytochrome P-450-CAM and myoglobin. Ultraviolet-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic investigation of the complexes. J Biol Chem 257 8308-8320... 

Liu HI, Sono M, Kadkhodayan S, Hager LR Hedman B, Hodgson KO, Dawson JH (1995) X-ray absorption near edge studies of cytochrome P-450-cam, chlo-roperoxidase, and myoglobin. Direct evidence for the electron releasing character of a cysteine thiolate proximal ligand. J Biol Chem 270 10544-10550... 

Figure 8.5. A catalytic cycle for hydroxylation by cytochrome P-450-CAM. The first four intramediates have been identifi the n-cation radical is speculative.

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. 

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-... 

The oxidation of /-butyl methyl ether to /-butanol (Steffan et al. 1997) which is mediated by the cytochrome P-450 from camphor-grown P. putida CAM, but not by that from R. rhodochrous strain 116. 

FIGURE I Schematic alignment of the deduced amino acid sequences of nitric oxide synthases (NOSs) and the cytochrome P-450 reductase. Depicted are consensus binding sites for heme, L-arginine, calmodulin (CaM), flavin mononucleotide (FMN), flavin-adenine dinucleotide (FAD), and NADPH. An NH2-terminal myristoylation site (myr) is present only in the endothelial constitutive NOS (ecNOS). n. Neuronal i, inducible. 

Fig. 16. EXAFS spectra of oxygenated cytochrome P-450 at pH 7.4 (top) and oxygenated chloroperoxidase at pH 6.0 (bottom) obtained at — 80°C. A mixed solvent was employed consisting of ethylene glycol (65% v/v) and 0.035 M potassium phosphate buffer (plus 4mM camphor for P-450-CAM). Spectra have been multiplied by to enhance the visibility of oscillations at high k. Reproduced with permission from Ref. [116]...

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