Dinuclear site radical

Iron chelators (type 2) are of medical interest as agents that can block cell proliferation. To what extent this is due to interference with RNR is not well understood. However, recent experiments on the isolated proteins show that an agent such as desferrioxamine can destroy the radical and chelates iron from the active form of the HSVl protein 111), whereas it has no direct effect on the tyrosyl radical of mouse R2 112). However, it seems that the iron in the dinuclear sites without neighboring tyrosyl radical is accessible to desferrioxamine complex-ation in mouse as well as HSVl R2, again suggesting a structural difference between iron sites with and without a neighboring radical. 

We begin this overview of manganese biochemistry with a brief account of its role in the detoxification of free radicals, before considering the function of a dinuclear Mn(II) active site in the important eukaryotic urea cycle enzyme arginase. We then pass in review a few microbial Mn-containing enzymes involved in intermediary metabolism, and conclude with the very exciting recent results on the structure and function of the catalytic manganese cluster involved in the photosynthetic oxidation of water. 

In contrast to the active site of galactose oxidase, to pre-catalyst 13, and to the system reported by Stack et al., the proposed catalytic species 15 does not imdergo reduction to Cu intermediates, as the oxidation equivalents needed for the catalysis are provided for solely by the phenoxyl radical Hgands. Since the conversion of alcohols into aldehydes is a two-electron oxidation process, only a dinuclear Cu species with two phenoxyl ligands is thought to be active. Furthermore, concentrated H2O2 is formed as byproduct in the reaction instead of H2O, as in the system described by Marko et al. [159]. 

The first bona fide MnRNR, isolated from Corynebacterium (formerly Brevibacterium) ammoniagenes, was reported about 10 years ago [122], The MnRNR R1 protein is monomeric (dimeric in E. coli) [123]. A dinuclear Mnm—0—Mnm unit was believed to be present at its active site (cf. oxidized E. coli RNR), and its sensitivity to hydroxyurea (a radical scavenger) suggested a mechanism similar to that of its iron counterpart. However, no tyrosyl radical EPR signal was observed from the MnRNR, casting doubts about the redox role of the manganese center [118], Very recently, however, a stable EPR signal, which is inhibited by hydroxyurea and correlates directly with enzymatic activity, has been detected [124],... 

Of the diiron enzymes, the one from E. coli is the best characterized and consists of two components. Subunit B1 (aa, 70 kDa) contains the nucleotide binding site, the thiols required for the reduction, and several effector sites (22). Subunit B2 (p2> 87.5 kDa) contains the catalytically essential tyrosyl 122 radical (23) and the dinuclear iron site (24). Both subunits were cloned separately and overexpressed in E. coli, thereby affording enzyme of sufficient quantities for biophysical studies (25). 

The nature of other ligands in this dinuclear complex can be deduced from other spectroscopic data. It is clear that the tyrosyl radical is NOT a ligand. Reduction of the tyrosyl radical with hydroxyurea does not alter the features of the Mdssbauer (26) and EXAFS (40) data. Indeed, the only hint that the radical is in the vicinity of the dinuclear iron unit is the temperature dependence of the EPR relaxation properties of the radical signal. At liquid helium temperatures, the tyrosyl radical behaves like an isolated organic radical. But, at temperatures where the dinuclear iron complex becomes paramagnetic, the radical signal becomes harder to saturate relative to an isolated radical (114). Based on these effects, the radical is estimated to be within 10 A of the diferric site. 

E. coli has recently been solved (29) it confirms the presence of one dinuclear iron site per subunit, with each site being associated with its own tyrosine 122 radical. The diferric active site is shown in Fig. 3b. 

In RNR R2 it is thought that, along with the dinuclear Fe(II) pair and the formation of a tyrosyl radical, a third Fe(II) is oxidized by dioxygen enabling its reduction to water (see Chapter 16) [48]. This third Fe(II) may be in solution or at a third site on the protein of unknown position. In EcFtna the known third Fe site (C) is located on the inside surface of the protein shell at ca 6 A from the dinuclear center... 

A connection between the manganese catalyzed formation of benzyl radicals and tyrosyl radicals can be formally established. It is known that Mn can replace Fe in some ribonucleotide reductases (RR) with retention of activity. Also, a dinuclear Mn active site has been proposed for authentic manganese ribonucleotide reductases (MnRR).l Both RR s share the X-oxo bridged dinuclear manganese motif with 1, except that, as discussed above, 1 comprises two such structural units. 

The non-heme enzyme methane monooxygenase (MMO) from methanotropic bacteria catalyzes the hydroxylation of methane to methanol. Methane is most difficult to hydroxylate and cytochrome P-450 cannot perform this reaction. MMO consists of three components. Component A is a dimer with subunits of dinuclear iron with monooxygenase activity. Components B and C are electron donor and transfer sites. Like cytochrome P-450, a high valent iron-oxo complex is proposed for component A in MMO. This species abstracts a H atom from CH4 to generate a CHs" radical. 

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