Phenolase activity

The hydroperoxo complex of [Cu2(L66)] has been spectroscopically characterized at low temperature. Its spectral features consist of a moderately intense LMCT band at 342 nm (As 8600 M cm ) and two weaker bands at 444 nm (As 850M cm ) and 610 nm (As 400 M cm ). This pattern of LMCT bands differs from those exhibited by mononuclear copper(II)-hydroperoxo complexes and suggests a bridging binding mode of the 

[Cu2(XYL)] does not form a stable dioxygen adduct and undergoes a fast ligand hydroxylation, while the peroxo complex of [Cu2(L66)] is a reactive species that can be used towards exogenous substrates. 

Indeed, the oxygenation of 4-carbomethoxy-phenoIate by [Cu2(L66)] and [Cu2(L55)] was previously reported to occur also in acetonitrile at -40° C, where the reactions were normally carried out by exposing the Cu(I)-phenolate adduct to dioxygen 136). Since acetonitrile freezes above the temperature range of stability of the copper-peroxo complex, it could not be established whether the peroxo species binds and reacts directly with the phenolate in these systems. Moreover, when the phenol hydroxylation is carried out at room temperature, the main product obtained is the dimeric compound resulting from a formal Michael addition of the phenolate on 

Treatment of [Cu(L6Ph)], the mononuclear analogue of [Cu2(L66)] , with dioxygen at low temperature also produces a stable peroxo complex 140). Despite the oxygenation occurs to a partial extent, the reversibility of the process, the spectral features of the complex (LMCT bands at 356 nm, s 20,000 M cm , and 560 nm, of very weak intensity), and further evidence 

The reactions of neutral phenols with two distinct dicopper-dioxygen complexes, a p-q q -peroxodicopper(II) complex and a bis(p-oxo)dicopper(III) complex, have been recently compared in a systematic study using the copper(I) complexes with the two related ligands LPy2 and LPyl (141). The Cu(I) complex with LPy2 yields predominantly the -peroxo 

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Fukuzumi and Itoh have jointly reported on a if-peroxo dicop-per(ll) complex that acts as a functional model for the phenolase activity of tyrosinase. lithium salts of para-substituted phenols were used as substrates, reaching yields between 60 and 90% with only the catechol product formed... 

Catechol as an Activator of Tyrosinase. The phenolase activity of tyrosinase has been studied less completely than the catecholase activity, partly because of the lack of a satisfactory assay procedure. The phenolase reaction, however, is characterized by a lag time which can be abolished by adding dihydroxyphenylalanine (DOPA), the immediate product of the hydroxylation reaction 29S4, 102, 117), This phenomenon has been described by several investigators (29-34) and is illustrated in Figure 12, from Pomerantz and Warner (117), using the enzyme from Hamster melanoma. The same phenomenon has been analyzed by Duckworth and Coleman (102) for the mushroom enzyme. In the absence of DOPA, maximum velocity of the hydroxylase reaction is not reached for several minutes. Pomerantz and Warner (117) devised a convenient assay for the phenolase reaction by determining the radio-... 

Some enzymes capable of oxidizing C-H bonds contain copper ions [52]. For example, tyrosinase [53a] contains a coupled, binuclear copper active site which reversibly binds dioxygen as a peroxide that bridges between the two copper ions. This enzyme catalyzes the orthohydroxylation of phenols with further oxidation of catechol to an ortho-quimne [53b-d], The mechanism proposed for phenolase activity of tyrosinase is shown in Scheme XI. 13 [521]. 

Dioxygen activation is also accomplished at the dinuclear copper-active sites in tyrosinases and catechol oxidases. Tyrosinases (EC 1.14.18.1) are widely distributed throughout bacteria, fungi, plants, and animals, catalyzing the ortho-hydroxylation of phenols to catechols (phenolase activity. Equation (1)) and the oxidation of catechols to o-quinones (catecholase activity. Equation (2)). 

Many chemists have been fascinated by metalloenzyme-catalyzed oxygenation of various organic substrates with molecular oxygen. The phenolase activity of tyrosinase is one such reaction. Thus, great efforts have so far been made to develop oxygenation reactions of phenols by molecular oxygen catalyzed by various copper complexes. However, little information is available about the mechanistic details of such catalytic oxidation reactions. 

Tyrosinase. Tyrosinase is a very well-studied multicopper oxygenase, which contains a magnetically coupled dinuclear copper center. This enzyme catalyzes the ortho-hydroxylation of phenols to catechols (phenolase activity, also called cresolase activity), as well as the 2-electron oxidation of catechols to 0-quinones (catecholase activity), which is the initial step for the biosynthesis of melanin (Scheme 1) (7,13). 

The involvement of phenols and enzymes of the phenolase complex appears to be secondary to the induction of necrosis. The induction must involve a modification of membrane structure which leads to altered membrane permeability and loss of cell compart-mentalization. If this occurs, regulation of cellular metabolism is lost, enz3mies are activated, and these and their substrates that are normally separated by membranes would react together. 

The presence of oxygen was shown to enhance inactivation of the enzyme, while the presence of ovalbumin in the phenolase solution protected the activity. This was accepted as evidence that indirect action of the radiation was responsible for the inactivation of phenolase since the presence of dissolved oxygen or protein would have no effect on direct collisions of the 7-rays and the enzyme molecules. 

Figure 10-13 Phenolase Catalyzed Reactions. (A) activation of phenolase. (B,C,D) Two-step four-electron reduction of oxycuprophenolase, and the associated hydroxylation of monophenols. Source From H.S. Mason, Mechanisms of Oxygen Metabolism, Adv. Enzymol., Vol. 19, pp. 79-233, 1957.

The dominant feature of tyrosinase is that it has both cresolase and catecholase activity. Laccase has a very clear catecholase activity, but its cresolase activity is not so clear. Mason, Fowlks, and Peterson (109) used 02 to label 3,4-dimethylphenol during the tyrosinase-catalyzed oxidation of this compound and showed that the source of the oxygen introduced into the phenol in the phenolase reaction was molecular oxygen according to Reaction 1. 

Mechanistic aspects of the action of tyrosinase and the usual transduction schemes have been summarized on several occasions [166,170-173]. In short, this copper enzyme possesses two activities, mono- and di-phenolase. Due to the predominant presence of the mono phenolase inactive form (met-form), the enzyme is inherently inefficient for the catalysis of these monophenol derivatives. However, in the presence of a diphenol, the catalytic cycle is activated to produce quinones and the scheme results in an efficient biorecognition cascade. This activation is achieved more efficiently when combined with electrochemical detection through the reduction of the produced quinones [166], as illustrated in Fig. 10.5. Consequently, a change in the rate-hmiting step can be observed through kinetic to diffusion controlled sensors with a concomitant increase in stability and sensitivity, as depicted in Fig. 10.6. 

As compared to the oxygenation reaction of phenols to catechols (phenolase reaction), dehydrogenation of catechols to the corresponding o-quinones (catecholase reaction) proceeds more readily. Thus, the catalytic activity of several tyrosinase and catechol oxidase models have been examined using 2,4-di-tert-butylcatechol (DTBC) as a substrate.Direct reactions between the (/r-77 77 -peroxo)dicopper(II) complexes and DTBC also have been studied at a low tempera-and a semiquinone-copper(II) complex has been isolated and structurally characterized... 

In this chapter, the dioxygen activation mechanism at the dinuclear copper-active sites of tyrosinase and catechol oxidase has been surveyed. In both enzymes, a (ji-rfirf -peToxo) dicopper(II) complex has been detected and characterized as a common reactive intermediate by several spectroscopic methods. In spite of longstanding efforts in the enzymological studies, mechanistic details of the enzymatic reactions (phenolase and catecholase activities) still remain ambiguous. On the other hand, recent developments in the model chemistry have provided a great deal of information about the structure and physicochemical properties as well as the reactivity of the peroxo intermediate and have advanced our understanding of the enzymatic reactions. 

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