Hydroxylases catalytic cycle

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.

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). 

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],...

Xanthine oxidase and related hydroxylase enzymes exhibit broad substrate specificity and an apparently complex catalytic cycle (109, 237). The important centers identified by EXAFS and EPR studies have... 

Figure 1 Catalytic cycle of p-hydroxybenzoate hydroxylase. In the first step, pOHB and NADPH bind (k- ) and the FAD becomes reduced (l<2). NADP is released (l<3) and O2 reacts (l<4) to form the C4a-hydroperoxy-FAD (E FIHOOH-S) in complex with substrate. Hydroxylation occurs via l<5 to yield the dienone form of product and the C4a-hydroxy-FAD (Int II). Tautomerization yields 3,4-dihydroxybenzoate in complex with the enzyme (E FI HOH-P). Dissociation of 3,4-DOHB and H2O via ky leads to free enzyme (E Flox). Uncoupling occurs via the loss of H2O2 from the C4a-hydropeoxy flavin (kg).

Tyrosine monooxygenase uses biopterin as a cofactor. Biopterin is made in the body and is not a vitamin. Its structure resembles that of folic acid. Dopa decarboxylase is a vitamin B -requiring enzyme. Dopamine hydroxylase is a copper metalloenzyme. The active form of the enzyme contains copper in the reduced state (cuprous, Cu+). With each catalytic event, the copper is oxidized to the cupric state (Cu ). The enzyme uses ascorbic acid as a cofactor for converting the cupric copper back to cuprous copper. Thus, each catalytic event also results in the conversion of ascorbic acid to semidehydroascorbate. The semidehydroascorbate, perhaps by disproportionation, is converted to ascorbate and dehydroascorbate. The catalytic cycle of dopamine hydroxylase is shown in Figure 9,86. Dopamine hydroxylase, as well as the stored catecholamines, are located in special vesicles... 

Experimental studies [16] show that the best-characterized forms of the soluble MMO (sMMO) contain three protein components hydroxylase (MMOH), so-called B component (MMOB) and reductase (MMOR), each of which is required for efficient substrate hydroxylation coupled to NADH oxidation. The hydroxylase, MMOH, which binds O2 and substrate and catalyzes the oxidation, is a hydroxyl-bridged binuclear iron cluster. In the resting state of MMOH (MMOHqx), the diiron cluster is in the diferric state [Fe -Fe ], and can accept one or two electrons to generate the mixed-valence [Fe -Fe ] or diferrous state [Fe -Fe ], respectively. The diferrous state of hydroxylase (MMOHred) is the only one capable of reacting with dioxygen and initiating the catalytic cycle. 

The iron of hydroxylases is at the active site and required for activity, but no evidence exists that this iron transfers electrons or binds oxygen during the catalytic cycle. At this stage, tetrahydropterin 4a-hydroperoxide is favored as the common intermediate responsible for hydroxylation in both mammalian and bacterial systems [122]. 

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