Mechanisms hydroxylation

G.S. Boyd u. R.M.S. Smellic, Biological Hydroxylation Mechanisms, Academic Press, London New York... 

Monooxygenase Protein System Structure, Component Interactions, and Hydroxylation Mechanism Katherine E. Liu and Stephen J. Lippard... 

STUDIES OF THE SOLUBLE METHANE MONOOXYGENASE PROTEIN SYSTEM STRUCTURE, COMPONENT INTERACTIONS, AND HYDROXYLATION MECHANISM... 

Since work with the radical clock substrate probes indicated important differences in the hydroxylation mechanisms for M. capsulatus (Bath) and M. trickosporium OB3b, work with (R) and (S)-[1-2H,1-3H]ethane with both enzymes was carried out (93, 94). With M. tri-chosporium OB3b, approximately 65% of the product displays retention of stereochemistry (93). A rebound rate constant of 2 - 6 x 1012 s-1 was calculated, assuming a free energy change of 0.5 kcal mole-1 for rotation about the C-C bond (94). This estimate approaches the value obtained from the radical clock substrate probe analysis (59). 

Physical studies of the hydroxylase have established the structural nature of the diiron core in its three oxidation states, Hox, Hmv, and Hred. Although the active site structures of hydroxylase from M. tri-chosporium OB3b and M. capsulatus (Bath) are similar, some important differences are observed for other features of the two MMO systems. The interactions with the other components, protein B and reductase, vary substantially. More structural information is necessary to understand how each of the components affects the others with respect to its physical properties and role in the hydroxylation mechanism and to reconcile the different properties seen in the two MMO systems. The kinetic behavior of intermediates in the hydroxylation reaction cycle and the physical parameters of intermediate Q appear similar. The reaction of Q with substrate, however, varies. The participation of radical intermediates is better established with the M. triehosporium... 

B3b system, although it certainly is not ruled out for the M. capsulatus (Bath) enzyme. In comparison to the cytochrome P-450 system, the hydroxylation mechanism for both MMO systems either has a rebound rate constant which is much larger and/or it takes place by an alternative pathway to classical radical rebound. 

In addition to a well-known NADPH-dependent hydroxylation mechanism (Reaction (2)), cytochrome P-450 is able to catalyze the oxidation of substrates by peroxygenase mechanism (Reaction (8)) where XOOH presents the peroxy compound acting as the oxygen donor. 

In the framework of these ideas, the phenol hydroxylation mechanism is implemented by oxygen transfer from the oxaziridine intermediate to phenol giving different intermediate compounds—benzene epoxide ... 

Another idea, theoretically justified by Goddare [131], about the hydroxylation mechanism of phenol compounds is based on the suggestion that the active intermediate compound is presented by biradical particles. 

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+. 

The kinetic regularities observed lead to consideration of the hydroxylation mechanism in the context of modem ideas about enzymatic catalysis mechanism. 

The study of acidic sites on aluminum-magnesium silicate carrier has detected weak (Broensted) and moderate (Lewis) sites with thermal desorption maxima at Tmax = 200 °C and Tmax = 400 °C, respectively. The mimic PPFe3+OH/AlSiMg derived on the basis of this carrier possesses weak sites only at 7 rnax = 250 °C. The absence of moderate acidic sites in the mimic indicates hematin adsorption from these mere sites, whereas weak acidic sites of the carrier participate in the hydroxylation mechanism. 

Although regulation is unique for each member of the aromatic amino acid hydroxylase family, the catalytic mechanism and cofactor requirements for members of the family are identical. During the reactions of all three enzymes, the dioxygen molecule is cleaved and incorporated as a hydroxyl group into both the aromatic amino acid and BH4. Each enzyme in the family displays its own unique substrate specificity profile. Two interesting questions about this enzyme family relate to the actual hydroxylation mechanism and how enzyme activity is altered by changes in BH4 levels. Problems in any one of these hydroxylation systems can arise from either an inadequate supply of the BH4 cofactor or a defect in the enzyme or its expression. 

As for iron, there is ample evidence that OH is generated in the Fenton reaction when the latter is carried out at acidic pH, but direct OH generation is often questionable at neutral or basic pH [4,24]. As above mentioned, other hydroxylation mechanisms have been proposed, based on the formation of hyper-valent iron species [100] such as perferryl (Fe=03+) or ferryl (Fe=02+), whose oxidizing powers are smaller than that of OH, for example ... 

Little evidence is available, therefore, that reductants induce redistribution of electronic charge within the prosthetic group in such a manner that oxygen of EO becomes the locus of the nucleophilic attack. The best reductants of Compound I are hard donors rather than polarizable substrates (cyanide, thiols, carbon monoxide, and isonitriles)—observations which cannot be readily reconciled with the hydroxylation mechanisms... 

Studies with cell-free hydroxylases suggest that the hydroxylation mechanisms are complex. It is assumed that an electron transport system involving an NADPH-dependent flavoprotein, an iron-sulfur protein, and cytochrome P-450 is involved. In the case of the steroid 15/S-hydroxylase system of Bacillus megaierium, these three components have been demonstrated15. The 1 la-hydroxylase of Rhizopus nigricans is also an enzyme of the P-450 monooxygenase type which works with an NADPH-cytochrome P-450 reductase. In this case the enzyme complex is associated with the endoplasmic reticulum of the mycelial cells34. 

Little is known of the active species and the hydroxylation mechanism, ft is even unclear whether the mechanism is homolytic or heterolytic. Accordingly, mechanisms of both types have been proposed (Schemes 18.4 and 18.5). 

---