In ribonucleotide reductase

From these data it seems feasible that a Co(II)-species is generated during catalysis, and that homolysis of the Co—C-bond is a prerequisite for enzyme catalysis in ribonucleotide reductase. However, the kinetics of appearance of the Co(II)-signal indicates that the rate of formation of Co(II) is much slower than either the rate of ribonucleotide reduction... 

Bar, G., M. Bennati et al. (2001). High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy The tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast. J. Am. Chem. Soc. 123 3569-3576. 

Un, S., M. Atta et al. (1995). g-values as a probe of the local protein environment High-field EPR of tyrosyl radicals in ribonucleotide reductase and photosystem II. J. Am. Chem. Soc. 117 10713-10719. 

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. 

Magnesium is bound in the active site of D-xylose isomerase (Carrell et al., 1984, 1989 Farber et al., 1987 Key etal., 1988 Henrick a/., 1989). Here, the site at which two metals [from among Mg(II), Mn(ll), and Co(II)] bind is similar to that found for Fe(ll) in ribonucleotide reductase (Nordlund et al., 1990). The active site of xylose isomerase is shown in Fig. 28. Magnesium ions are preferred in ATP-utilizing enzyme reactions (Mildvan, 1987). 

Fig. 13. Environment of the binuclear iron in ribonucleotide reductase. (Reproduced with permission from Nordlund et al., 1990.)...

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.

Stubbe, J. Riggs-Gelasco, P. (1998) Harnessing free radicals formation and function of the tyrosyl radical in ribonucleotide reductase. Trends Biochem. Sci. 23, 438-443. 

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. 

Denysenkov, V. P., Prisner, T. F., Stubbe, J., and Bennati, M. (2006). High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 103, 13386-13390. 

Mitic N, Saleh L, Schenk G, Bollinger JM, Solomon El. Rapid-freeze-quench magnetic circular dichroism of intermediate X in ribonucleotide reductase new structural insight. J Am Chem Soc. 2003 125 11200-1. 

Siegbahn PEM, Eriksson L, Himo F, Pavlov M. Hydrogen Atom Transfer in Ribonucleotide Reductase (RNR). J Phys Chem B. 1998 102 10622-9. 

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... 

The generation, stability, and function of tyrosyl radicals in ribonucleotide reductase, PGH synthase, and galactose oxidase continue to be active areas of research. The difficulties encountered in preparing and handling these proteins, as well as in probing the physical properties and reactivity of their metal-phenoxyl radical active sites, make the preparation and investigation of stable phenoxyl radical metal model complexes an attractive goal. 

Fig. 9. Redox-active amino acid residues related to tyrosine, (a) Tyrosine, the redox center in ribonucleotide reductase, prostaglandin H synthase, and the photosynthetic oxygen evolving complex, (b) 2,4,5-Trihydroxyphenylalanine, the redox cofactor of the quinoprotein amine oxidase, (c) Tyrosine-cysteine (Tyr-Cys), the redox cofactor of galactose oxidase.

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. 

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