Tyrosine radical, in ribonucleotide

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.

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]. 

Bleifnss G, Kolherg M, Potsch S, Hofbauer W, Bittl R, Luhitz W, Graslimd A, Lass-mann G, Lendzian F. 2001. Tryptophan and tyrosine radicals in ribonucleotide reductase A comparative high-field EPR smdy at 94 GHz. Biochemistry 40 15362—15368. 

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. 

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. 

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. 

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.

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

Figure 7. ESR spectra ( —196°C) of tyrosyl radicals in ribonucleotide reductase from E. coli. a, Spectrum from enzyme grown in the presence of tyrosine b, spectrum from enzyme grown in the presence of deuterated [yJ./i- HjJtyrosine. From [137], with permission.

Fig. 2. EPR spectra of tyrosine radicals (A) the stable tyrosine radical, Tyr[,-, in photosystem II (65 ij,M) in a frozen solution containing 30% (v/v) ethylene glycol in water and (B) the stable tyrosine radical in the B2 subunit of Escherichia coli ribonucleotide reductase (50 jtiAf) in a frozen solution containing 10% (v/v) ethylene glycol in water. Conditions temperature, 8.0 K microwave frequency, 9.05 GHz magnetic field modulation amplitude, 2 G magnetic field modulation frequency, 100 kHz microwave power, 0.7 /iW.

Fig. 5. Progressive microwave power saturation plot of the tyrosine radical in the B2 subunit of E. coli ribonucleotide reductase. Conditions were as in Fig. 2B.

Fig. 3a-c. ESR spectra of the tyrosine free radicals in ribonucleotide reductases, a protein subunit B 2 of . coli enzyme, measured at 86 K "> b bacteriophage T4 enzyme, at 77 K c hydroxyurea-resistant, reductase-overproducing mouse 3T6 cells, at 32 K ... 

The main source of thiyl radicals in cells is expected to be reaction (1), because of the relative abundance of C-H bonds. Some biological radicals are nitrogen-or oxygen-centered, such as the tryptophan (indolyl) radical in DNA photolyase [45], and indolyl and tyrosine (phenoxyl) radicals in ribonucleotide reductase [46-49]. Whether oxygen-centred radicals such as phenoxyl radicals (PhO, e.g. from tyrosine) oxidize GSH by hydrogen transfer ... 

Recent EPR studies have shown that the amino acid tyrosine participates in a number of biological electron transfer reactions, including the oxidation of water to Oj in plant photosystem II, the reduction of Oj to water in cytochrome c oxidase, and the reduction of ribonucleotides to deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase. During the course of these electron transfer reactions, a tyrosine radical forms (4). The center of the EPR spectrum of the tyrosine radical in cytochrome c oxidase of the bacterium P. denitrificans occurs at 344.50 mT in a spectrometer operating at 9.6699 GHz (radiation belonging to the X band of the microwave region). Its -value is therefore... 

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.)...

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... 

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. 

The iron protein has been found in animals, viruses and some prokaryotic organisms. The best studied example is from Escherichia coli.S19 The enzyme consists of two subunits, Bi and B2. Subunit B, has a molecular weight of 160 000 and is made up of two polypeptide chains. It contains redox-active SH groups, which play a role in the reaction, and has two binding sites for substrates and sites for effector molecules. It can bind any of the four ribonucleotide substrates. Subunit B2 has a molecular weight of 78 000 and also has two polypeptide chains. It contains a dimeric oxo-bridged iron site associated with a remarkably stable tyrosine radical. The formation of the active enzyme from B, and B2 requires the presence of magnesium ions. 

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... 

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. 

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