Hydroxylase cycle

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein FejSj. Carbon monoxide (CO) inhibits the indicated step.

Although /3-oxidation is universally important, there are some instances in which it cannot operate effectively. For example, branched-chain fatty acids with alkyl branches at odd-numbered carbons are not effective substrates for /3-oxidation. For such species, a-oxidation is a useful alternative. Consider phy-tol, a breakdown product of chlorophyll that occurs in the fat of ruminant animals such as sheep and cows and also in dairy products. Ruminants oxidize phytol to phytanic acid, and digestion of phytanic acid in dairy products is thus an important dietary consideration for humans. The methyl group at C-3 will block /3-oxidation, but, as shown in Figure 24.26, phytanic acid a-hydroxylase places an —OFI group at the a-carbon, and phytanic acid a-oxidase decar-boxylates it to yield pristanie add. The CoA ester of this metabolite can undergo /3-oxidation in the normal manner. The terminal product, isobutyryl-CoA, can be sent into the TCA cycle by conversion to succinyl-CoA. 

Fig. 4. Proposed catalytic cycle for the hydroxylation of methane by MMO. The reductase and B components may interact with the hydroxylase in one or more steps of the cycle. Protons are shown in the step in which intermediate Q is generated, but other possibilities exist (see Fig. 3 and the text). The curved line represents a bridging glutamate carboxylate ligand.

Fig. 4. Substrate first binds to the complete system containing all three protein components. Addition of NADH next effects two-electron reduction of the hydroxylase from the oxidized Fe(III)Fe(III) to the fully reduced Fe(II)Fe(II) form, bypassing the inactive Fe(II)Fe(III) state. The fully reduced hydroxylase then reacts with dioxygen in a two-electron step to form the first known intermediate, a diiron(III) peroxo complex. The possibility that this species itself is sufficiently activated to carry out the hydroxylation reaction for some substrates cannot be ruled out. The peroxo intermediate is then converted to Q as shown in Fig. 3. Substrate reacts with Q, and product is released with concomitant formation of the diiron(III) form of the hydroxylase, which enters another cycle in the catalysis.

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... 

The NADH-dependent reductase, which contains a 2Fe 2S cluster and FAD as cofactors, converts the oxidized hydroxylase binuclear cluster to a diferrous state after each catalytic cycle. It should be emphasized that the reductase does not participate directly in the hydroxylation reaction its sole function is to regenerate the reduced enzyme in a separate reaction (Fox et al., 1988). The latter reacdon is reminiscent of the NADH-linked reducdon of inactive diferric RNRB2 (see Section III,B). 

Fluoro amino acids have been incorporated into peptides, in order to ease the transport or reduce the systemic toxicity. Thus, trifluoroalanine, a powerful inhibitor of alanine racemase, is an essential enzyme for the biosynthesis of the cell wall of bacteria. It has a low antibiotic activity because of its very poor transport. In order to facilitate this transport, the amino acid has been incorporated into a peptide. This delivery allows a reduction of the doses, and thus the toxicity of the treatment is lowered.3-FIuorophenylaIanine (3-F-Phe) is a substrate of phenylalanine hydroxylase, which transforms it into 3-F-Tyr. 3-F-Tyr has a high toxicity for animals, due to its ultimate metabolization into fluorocitrate, a powerful inhibitor of the Krebs cycle (cf. Chapter 7). 3-F-Phe has a low toxicicity toward fungus cells, but when delivered as a tripeptide 3-F-Phe becomes an efficient inhibitor of the growth of Candida albicans. This tripeptide goes into the cell by means of the active transport system of peptides, where the peptidases set free the 3-F-Phe. ... 

After removal of their amino groups, the carbon skeletons of amino acids undergo oxidation to compounds that can enter the citric acid cycle for oxidation to C02 and H20. The reactions of these pathways require a number of cofactors, including tetrahydrofolate and 5-adenosylmethionine in one-carbon transfer reactions and tetrahydrobiopterin in the oxidation of phenylalanine by phenylalanine hydroxylase. 

Figure 3. The catalytic cycle of soluble MMO. Oxygen atoms derived from molecular oxygen are shown in bold. Compounds P, P, Q, R, and Tare described in the text. MMOR, reductase component of MMO MMOH, hydroxylase component of MMO. Adapted from [68, 79],...

First two complexes with a (p.-oxo)(p-hydroxo)diiron (III) core [Fe2(0)(0H)(6TLA)2(C104)3] (I) and [Fe2(0)2(6TLA)2(C104)2] (II), were isolated and characterized (Zang et al 1995). Structure of a (-l,2-peroxo)bis(-carboxylato)diiron(m)model for the peroxo intermediate in the methane monooxygenase hydroxylase reaction cycle is presented in Fig, 6.3. 

Kim, K., and Lippard, S. J., 1996, Structure and M ssbauer spectrum of a (p-l,2-peroxo)-bis( r-carboxylato)diiron(III) model for the peroxo intermediate in the methane monooxygenase hydroxylase reaction cycle, J. Am. Chem. Soc. 118 4914n4915. 

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