Tyrosyl radical formation, ribonucleotide reductase

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. 

Bollinger Jr JM, Stubbe J, Huynh BH, Edmondson DE. 1991. Novel diferric radical intermediate responsible for tyrosyl radical formation in assembly of the cofactor of ribonucleotide reductase. JAm Chem Soc 113 6289-6291. 

The realization of the widespread occurrence of amino acid radicals in enzyme catalysis is recent and has been documented in several reviews (52-61). Among the catalytically essential redox-active amino acids glycyl [e.g., anaerobic class III ribonucleotide reductase (62) and pyruvate formate lyase (63-65)], tryptophanyl [e.g., cytochrome peroxidase (66-68)], cysteinyl [class I and II ribonucleotide reductase (60)], tyrosyl [e.g., class I ribonucleotide reductase (69-71), photosystem II (72, 73), prostaglandin H synthase (74-78)], and modified tyrosyl [e.g., cytochrome c oxidase (79, 80), galactose oxidase (81), glyoxal oxidase (82)] are the most prevalent. The redox potentials of these protein residues are well within the realm of those achievable by biological oxidants. These redox enzymes have emerged as a distinct class of proteins of considerable interest and research activity. 

Dinuclear iron centres occur in several proteins. They either bind or activate dioxygen or they are hydrolases. Ribonucleotide reductase (RR) of the so-called class I type contains one such centre in the R2 protein in combination with a tyrosyl radical, both being essential for enzymatic activity which takes place in the R1 protein subunit. The diiron centre activates dioxygen to generate the tyrosyl radicals which in turn initiate the catalytic reaction in the R1 subunit. The interplay between the tyrosyl free radical in R2 and the formation of deoxyribonucleotides in R1 which also is proposed to involve a protein backbone radical is a topic of lively interest at present but is outside the scope of this review. Only a few recent references dealing with this aspect are mentioned without any further discussion.158 159 1 1,161... 

Hydroxyurea is a simple molecule (Fig. 15-17) which inhibits ribonucleotide reductase. This enzyme accepts the four NDPs, UDP, CDP, ADP and GDP, as substrates and reduces them to the corresponding dNDP. The mechanism of catalysis involves the formation of an unusual tyrosyl radical cation which then induces formation of a radical form of the NDP substrate. Hydroxyurea quenches this tyrosyl radical cation intermediate leading to depletion of all four dNTPs required as substrates for the synthesis of DNA. 

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-... 

Ochiai, E., Mann, G. J., Gr%oslund, A., and Thelander, L., 1990, Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase. J. Biol. Chem. 265 15758915761. 

Structure of the Iron Center Formation of the Iron Center and Tyrosyl Radical Spectroscopy of the Diferric Iron Center Spectroscopy of the Tyrosyl Radical Redox Properties of the Iron Center Mixed-Valent Form of the Iron Center Diferrous Form of the Iron Center Inhibitors to Iron-Containing Ribonucleotide Reductase Methane Monooxygenase A. Spectroscopy of the MMOH Cluster X-Ray Structure of MMOH... 

Spectroscopic data support location of the radical in GAO on the Y272-C228 unit. The first indication came from UV-vis, EPR, and ENDOR studies of a one-electron oxidized form of Cu-depleted (apo) GAO, which showed the formation of a thioether-modified tyrosyl radical. " " This radical was found to be quite stable, as reflected by the oxidation potential of about-1-0.4 V (vs. normal hydrogen electrode (NHE)) which is significantly less than that of other tyrosine/ tyrosyl radical couples (cf.+0.93V for free tyrosine or +1.0V for the tyrosyl residue near the diiron site in ribonucleotide reductase, vide infra). Possible origins of this unusual stability that have been considered are the thioether substituent," the nearby W290, and/or other unspecified protein environmental effects. Coordination of this radical to Cu was then proposed for the... 

The role of oxygen in eukaryotic DNA biosynthesis may indeed be a critical one. It has recently been shown that O2 is not only required for initial formation of tyrosyl radical but must be continously present to maintain the radical content and enzyme activity of mammalian ribonucleotide reductase In vivo studies with Ehrlich ascites cells also point to a tight link between oxygen and deoxyribonucleotide supply Anaerobic arrest of cells in G1 phase and block of DNA synthesis can be relieved by addition of deoxycytidine, but not cytidine, to the culture medium. ... 

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. 

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