Hydroxylation with cytochrome

Zehentgruber D, Hannemann F, Bleif S, Bernhardt R, Lutz S (2010) Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2. Chembiochem 11 713-721... 

SCHEME 32.20 Selected examples of arene hydroxylation with cytochromes P450. 

With AT. trichosporium 0B3b, epimerization with exo, exo, exo, exo-2,3,5,6-d4-norbornane upon hydroxylation occurs (83), which parallels results for cytochrome P-450 hydroxylation with this substrate (91). The extent of epimerization with MMO, however, was significantly lower, being 2% following hydrogen atom abstraction from the endo position compared to 18% with cytochrome P-450, and 5% after abstraction at the exo position as compared to 14% with cytochrome P-450... 

Allylic rearrangements with 3,3,6,6-dj-cyclohexene occurred in 20% of the MMO hydroxylation products compared to 33% for cytochrome P-450. These two experiments suggest that, with M. trichospor-ium OB3b, a rebound reaction must occur with a greater rate constant than with cytochrome P-450, in accord with the radical clock substrate work. 

Fig. 14.8 Experimental ligand interactions with cytochrome P450 2C family. (A) X-ray structure ofthe sulfaphenazole derivate DMZ in rabbit CYP2C5 at 2.3 A resolution (PDB 1 N5B) from Wester et al. [191]. Only one ofthe two-ligand orientations for DMZ in accord with electron density is shown placing the benzylic methyl group in a 4.4 A distance to the heme iron. (B) X-ray structure of S-warfarin in human CYP2C9 at 2.55 A resolution (PDB 10G5) from Williams et al. [192]. The substrate is situated in a predominantly hydrophobic pocket. This binding mode places the 6- and 7-hydroxylation sites 10 A from the iron (arrow).

Iodosylbenzene has been extensively utilized over the last few years for its ability to cleanly transfer oxene oxygen atoms to metals and possibly generate high-valent reactive metal-oxo species. PhIO has been successfully used instead of 02+NADPH in conjunction with cytochrome P-450 to hydroxylate alkanes,81 and has found a variety of interesting applications as a two-electron oxidant in the presence of first-row transition metal porphyrins. 

D KIE associated with alkane hydroxylation by cytochromes P-450 and intermolec-ular D KIE in the alkane hydroxylations catalysed by manganese and iron porphyrin complexes... 

The most ambiguous point in the oxynoid hydroxylation mechanism with cytochrome P-450 is the question about the formation and origin of the oxygen intermediate Fe3+ O, where the oxygen atom (possessing six electrons in the external cover) is coordinated withFe3+. 

Harris, N., Cohen, S., Filatov, M., Ogliaro, F., and Shaik, S. (2000) Two-state reactivity in the rebound step of alkane hydroxylation by cytochrome P-450 origins of free radicals with finite lifetimes. Angew. Chem., Int. Ed. 39, 2003-2007. 

One motivation for the characterization of the above compounds has been to more fully understand the involvement of such higher valent manganese porphyrin complexes in model systems which imitate the catalytic activity of monooxygenase cytochrome P-450 and related enzymes. The catalytic cycle of cytochrome P-450 appears to involve the binding and reduction of molecular oxygen at a haem centre followed by the ultimate formation of a reactive iron oxo complex which is responsible for oxidation of the substrate. For example, cytochrome P-450 is able to catalyse alkane hydroxylation with great selectivity. 

Three alternative mechanisms were proposed based only on the thermodynamic data (403). All of these assumed distinct functions for each flavin and interaction between the flavins. They also assumed that electrons would be transferred to cytochrome P-450 one at a time this has been shown to be the case with cytochromes P-450 that receive electoons from iron-sulfur proteins rather than from the flavoprotein directly (or through the indirect mediation of lipid) (405, 406). One of these mechanisms (403) is shown below. It seems to fit best with the kinetic data determined for the lipase-solubilized reductase (345, 398). In this scheme, SH is a hydroxylatable substrate and SOH its hydroxylated product, and Fli and FU are the high potential and low potential flavins, respectively. 

Hydroxylation Hydroxylation can occur with an aliphatic or aromatic carbon atom. DDT and the carbamate insecticide carbaryl are known to be hydroxylated by cytochrome P450 monooxygenases as shown in Figures 8.3 and 8.4. Microsomal hydroxylation usually results in detoxification. 

W. Ando and Y. Moro-oka, Role of Oxygen in Chemistry and Biochemistry , Elsevier, Amsterdam, 1988. Includes chapters on (a) Cation-Radical Chain Catalyzed Oxygenation of Alkenes, by S. F. Nelsen, et al. (b) Photooxygenation of Organic Compounds via Thieir Radical Ions, by K. Mizono and Y. Otsuji (c) Triphenylpyrylium Sensitized Oxygenations, by K. Tokumaru a al. (d) Catalytic Oxidation of Metiiylarenes to Benzaldehydes, by R. A. Shddon and N. de Heij (e) Oxidation of Arylamines with Horseradish Peroxidase, 1 Fujimori et al. (f) Arene Hydroxylation by Cytochrome P-4S0, by M. Tsuda et al., and marry others. 

The biosynthesis of the ergot alkaloids involves condensation of dimethyl-allyl pyrophosphate (derived from mevalonic acid) with tryptophan. Closure of the C- and D-rings of the alkaloid involves specific hydroxylations by cytochrome P450-dependent oxidases and rearrangements. Further modifications involve N-methylation in the presence of S-adenosyl methionine and/or condensation with amino acids and peptides. Coupling of lysergyl CoA with certain peptides forms the peptide alkaloids which are the most bioactive of the ergot alkaloids. 

Shaik and co-workers have carried out a number studies using density functional theory based quantum chemical and QM/MM techniques to examine various aspects of the mechanism of alkane hydroxylation by cytochrome P450.178 181 These studies included, for example, calculation of the potential energy surface for the so-called rebound mechanism with methane as a substrate for two spin states, the high spin (HS) quartet state and low spin (LS) doublet state. In the rebound mechanism, Compound I initially abstracts a... 

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