Ribonucleotide reductase tyrosine radical

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

Sjoberg, B.-M., Reichard, P, Graslund, A., and Ehrenberg, A. 1978. The tyrosine free radical in ribonucleotide reductase from Escherichia coli. The Journal of Biological Chemistry 253 6863-6865. 

Langen, P., Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents an EPR study, Free Radical Biol. Med. 9 (1990), p. 1-4... 

In eukaryotes, ribonucleotide reductase is a tetramer consisting of two R1 and two R2 subunits. In addition to the disulfide bond mentioned, a tyrosine radical in the enzyme also participates in the reaction (2). It initially produces a substrate radical (3). This cleaves a water molecule and thereby becomes radical cation. Finally, the deoxyribose residue is produced by reduction, and the tyrosine radical is regenerated. 

Hydroxyurea selectively inhibits ribonucleotide reductase (see p. 190). As a radical scavenger, it removes the tyrosine radicals that are indispensable for the functioning of the reductase. 

Figure 6 High Field EPR spectra of radicals occurring in PS II ( Yz obtained at 245 GHz, all other spectra at 285 GHz). For comparison, the spectra of the tyrosine radical in ribonucleotide reductase (RNR) and of irradiated tyrosine hydrochloride crystals n are also shown (for details see reference 30). Note the striking differences in g values for the tyrosyl radicals. Figure reproduced from reference 30 with permission.

Figure 16-21 (A) Scheme showing the diiron center of the R2 subunit of E. coli ribonucleotide reductase. Included are the side chains of tyrosine 122, which loses an electron to form a radical, and of histidine 118, aspartate 237, and tryptophan 48. These side chains provide a pathway for radical transfer to the R1 subunit where the chain continues to tyrosines 738 and 737 and cysteine 429.354a c From Andersson et al.35ic (B) Schematic drawing of the active site region of the E. coli class IH ribonucleotide reductase with a plausible position for a model-built substrate molecule. Redrawn from Lenz and Giese373 with permission.

An unusual feature of ribonucleotide reductase is that the reaction it catalyzes involves a radical mechanism. The mammalian type of reductase initiates this reaction by the tyrosyl radical-nonheme iron. Hydroxyurea and related inhibit the majTrrrraiVarr retftrcCase 6y abolishing the radical state of the tyrosine residue. Inhibition of DNA synthesis by such compounds is secondary to this effect. 

The emphasis on the study of hemoproteins and the iron-sulfur proteins often distracts attention from other iron proteins where the iron is bound directly by the protein. A number of these proteins involve dimeric iron centres in which there is a bridging oxo group. These are found in hemerythrin (Section 62.1.12.3.7), the ribonucleotide reductases, uteroferrin and purple acid phosphatase. Another feature is the existence of a number of proteins in which the iron is bound by tyrosine ligands, such as the catechol dioxygenases (Section 62.1.12.10.1), uteroferrin and purple acid phosphatase, while a tyrosine radical is involved in ribonucleotide reductase. The catecholate siderophores also involve phenolic ligands (Section 62.1.11). Other relevant examples are transferrin and ferritin (Section 62.1.11). These iron proteins also often involve carboxylate and phosphate ligands. These proteins will be discussed in this section except for those relevant to other sections, as noted above. 

Fig. 5. Mechanism of action of dinuclear non-haem iron enzymes utilising ferryl intermediates. Mechanisms for ribonucleotide reductase and methane mono-oxygenase adapted from that of Que [72]. Compound I and compound II define intermediates at the same oxidation state as the equivalent peroxidase intermediate (see Fig. 2). X is an unknown group suggested to bridge between the two iron atoms and form a cation radical. The nature of the electron required for the reduction of ribonucleotide reductase compound II is not clear - it is possible that this intermediate can also oxidise tyrosine [72].

Unfortunately, despite the great stability of the tyrosine radical of ribonucleotide reductase [69], the crystals available for diffraction contain the enzyme in its non-radical form [107]. However, the tyrosine residue (Tyr-122) known to convert to the radical is buried in the protein [99] and the position and structure of the tyrosine residue in the crystal is consistent with spectroscopic data for the radical form as with cytochrome c peroxidase, it appears unlikely that major conformational changes occur subsequent to radical formation. 

However, recently it has proved possible to positively identify tryptophan radicals in cytochromec peroxidase[147] and tyrosine radicals in ribonucleotide reductase, prostaglandin H synthase and photosystem II of chloroplasts [148], This has been achieved by a combination of the techniques discussed already, but with the powerful, additional non-invasive tool of isotopic substitution. As deuterons (5=1) give different splitting than protons (S = 1/2), substituting different labelled amino-acid residues into the enzyme should reveal the nature of the radical-containing residue. This is easily achieved in an auxotrophic mutant that requires this amino acid to be supplied in the medium. The specific residue can then be identified by site-directed mutagenesis of the evolutionary conserved amino-acid residues [108,149-151]. 

However, it is possible to detect a tyrosine radical optically in ribonucleotide reductase, as there is only a relatively weak competing absorption from the binuclear non-haem iron centre [164]. A distinct sharp peak is seen that is not present in proteins that have been treated with the radical scavenger hydroxyurea [165,166] nor is it present in proteins such as haemerythrin or methane monooxygenase, which have similar active-site structures, but lack... 

The Mossbauer spectrum of native ribonucleotide reductase is very similar to that of oxyhaemerythrin, both having a binuclear Fein-Fein cluster with 5 = 0 [127,188]. This suggests that there is negligible coupling of the iron centre to the distant (5A) tyrosine-122 radical in ribonucleotide reductase. This was confirmed by the fact that removing the tyrosine radical with hydroxyurea or hydroxylamine had no effect on the spectrum [188]. However, by rapid-freezing... 

Fig. 9. Resonance Raman spectrum of tyrosine radical in purified E. coli ribonucleotide reductase (A) Pure B2 subunit (B) holoenzyme (C) metB2 subunit (lacking tyrosine radical). Note the correlation of the 1498 cm-1 band with the presence of the tyrosine radical. Reprinted with permission from Backes, G., Sahlin, M., Sjoberg, B.-M., Loehr, T.M. and Sanders-Loehr, J. (1989) Biochemistry 28, 1923-1929. Copyright 1989, American Chemical Society.

Free radicals also have distinctive Raman stretching frequencies. For example, ribonucleotide reductase (Fig. 9) has a stretch at 1498 cm-1 that is not present in the hydroxyurea-treated radical-free protein [208], This is close to that observed [209] for deprotonated phenoxy radicals (1505 cm-1), and different [210] from that of the protonated radicals (1426 cm-1). Thus it was concluded that the tyrosine radical was the neutral deprotonated radical, a conclusion that is difficult to reach from the EPR spectra alone. 

Ribonucleotide reductase catalyses the reduction of the four common ribonucleotides to their corresponding deoxyribonucleotides, an essential step in DNA synthesis. All four ribonucleotides are reduced by the same enzyme [77], The enzyme (250 000 mol. wt.) is a complex of two proteins Mi which contains substrate and redox-active sulphydryl groups and M2 which contains both a (x-oxo-bridged binuclear iron centre (Fig. 5) [77] and a tyrosine moiety sidechain which exists as a free radical stabilised by the iron centre [78], This radical, which is only 5.3 A away from iron centre 1, has access to the substrate-binding pocket and is essential for enzyme activity. Electrons for the reduction reaction are supplied from NADPH via thioredoxin, a small redox-active protein. 

The site in the active Fe ribonucleotide reductase contains two Fe(III) ions 3.3 A apart, bridged by one carboxylate from a glutamate residue and a water-derived oxo bridge (57). The function of this iron center appears to be the formation and stabilization of a free radical on a tyrosine about 5 A away. This radical is formed by reaction of the reduced, diferrous center with 02, probably through peroxide and ferryl intermediates. This unusually stable tyrosyl radical is thought to partic-... 

Reichard, 1972 Barry and Babcock, 1987). It is probably formed by electron-loss from the tyrosine side-group, followed by proton loss, since it is found in redox enzymes such as ribonucleotide reductase, which catalyses the conversion of ribonucleotides into deoxyribonu-cleotides. In this enzyme the tyrosyl is said to be stabilized by an adjacent binuclear iron unit, but the mode of stabilization is not clear, and the ESR and ENDOR spectra of the radical are not significantly perturbed, as would have been expected if any direct bonding were involved. 

This is markedly different from the behavior of a simple tyrosine phenoxyl, such as that found in ribonucleotide reductase, whose spectrum exhibits a strong rhombic splitting (Fig. 17, line c) but precisely the same as observed for the 0-methylthiocresyl model radical (Fig. 17, line b). This clearly identihes the Tyr-Cys side chain as the site of the oxidized apoGAOX radical and demonstrates that the electronic structure of the thioether-substituted phenoxyl is distinct from that of a simple phenoxyl radical. 

Larsson, A., and Sj"herg, B.-M., 1986, Identification of the stable free radical tyrosine residue in ribonucleotide reductase. EMBO J. 5 2037n2040. 

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