Enzyme complexes, tyrosyl radicals

The continuous development of phenoxyl radical complexes started with an aim of modeling primarily the enzyme GO. The number of papers cited testily to the uninterrupted interest in this chemistry. Thus a small selection of more recent references from the literature including reviews (224), theoretical (225) and model studies (226), and characterization of tyrosyl radicals in biological systems (227) will close this chapter. 

For nonheme enzymes that fiuther activate dioxygen, it is apparent that the diferrous forms also bind O2 to eventually generate the active species responsible for the oxidative transformations. In the case of MMO, the first intermediate has been labeled compound P (Scheme 2), which subsequently converts to compound Q both are kinetically competent to hydroxylate methane. In the case of RNR, compound X (Scheme 1) is responsible for the one-electron oxidation of a tyrosine residue to generate a tyrosyl radical. Based on chemical considerations and its Mossbauer properties, it has been proposed that compound P is a diferric peroxide species. To date, three model complexes of compound P, with comparable spectroscopic properties, have been structurally characterized (Figure g). In two of these models O2 is bound in a cis... 

Coordinated tyrosyl radicals have been discovered in the active form of the Cun-containing fungal enzyme, galactose oxidase. The unpaired electron on the ligand is strongly anti-ferromag-netically coupled to the unpaired d electron. Phenoxyl radical ligands have been confirmed in the successive one-electron oxidation of [Ga(L )3] by spectroelectrochemistry of the Ga model complex (Li shown in Scheme 2). 

The firee radical concentration does not significantly exceed 1 per B2 subunit even in most active and most concentrated enzyme samples Thus, the molecular composition of this reductase subunit can be viewed as 2 tightly aggregated polypeptide chains, 2 coupled iron atoms, and 1 radical, or under a more functional aspect as 1 native protein containing 1 binuclear iron complex and 1 tyrosyl radical (Fig. 2). 

The effects of hydroxyurea in the purified ribonucleotide reductase systems of E. coU, phage T4, calf thymus and mouse cells have been described above (p. 36, 42). Inhibition of substrate reduction in vitro (I50 = 2 - 3 10 ) is accompanied by loss of the tyrosyl radical, but not iron from E. coli subunit B2. Studies with substituted hydroxyl-amines and hydroxamates showed good correlation between their ability to undergo one-electron oxidation and enzyme inhibition, unless branched substituents prevented interaction with the protein (Table 8) Thus the mode of inhibition of E. coli ribonucleotide reductase is essentially solved Within steric restrictions of accessibility to the active site the compounds donate an electron to the enzyme s free radical, producing an inactive protein with still intact binuclear iron complex (Eq. VI). This process is irreversible in vitro until iron is removed, and then reintroduced with Fe(II)ascorbate in the presence of oxygen, whereupon radical and enzyme activity reappear. No other enzyme of E. coli has been found to be inactivated by hydroxyurea. 

The active site of this enzyme in its Cu(II) form contains fom amino acids, two tyrosins one of which is a tyrosyl radical, two histidines, and one solvent molecule that together form a five-coordinate metal complex (54,76). During the reduction-oxidation reactions, the coordination sphere changes. In the Cu(I) state, only three amino adds are coordinated to the central cation. The unusual two-electron oxidation reaction involves the reduction of the central Cu(II) cation and the tyrosyl radical. 

The distinctive feature of NO is the capability of forming weak reversible complexes with some stable free radicals. The formation of such complexes is characteristic of tyrosyl radicals identified in a number of enzyme systems [16]. The interaction of tyrosyl radicals with NO has been shown to inhibit enzyme activity. The reaction between photogenerated tyrosyl radicals and NO is near diffusion-limited with the rate constant of 1-2 x 10 /M/s and gives 3-nitrosocyclohexadienone intermediate species. These species can reversibly regenerate the tyrosyl radical and NO or undergo rearrangement to form 3-nitrosotyrosine ... 

Two phenoxyl radical complexes [Cu (2 )N03] and [Zn (2 )N03] oxidize benzyl alcohol to benzaldehyde and have been studied as models for the enzyme galactose oxidase (GO). GO contains a dipeptide unit (3) in which a tyrosine residue is covalently bound to an adjacent cysteine residue and which is similar to (2), the tyrosyl radical in (3) also being bound to the Cu centre (see Figure 1). Second-order kinetics were observed with respect to [Zn°(2 )N03]+ and there was no evidence of redox reaction at the zinc site, suggesting that a dimeric form of the complex is active however, the reaction of [Cu H2 )N03]+ with benzyl alcohol is first order in the metal complex and [Cu (2H)]+ is identified as a product, suggesting a formal 2e /2H+ mechanism in which the monomeric form coordinates the alcohol in the manner believed to operate for G0. 2... 

---