Ribonucleotide reductase tyrosyl radical stability

Nitric oxide is highly reactive toward other radicals. Thus, radical-radical addition is the most likely route for any potential direct effects of nitric oxide. Ribonucleotide reductase contains a stabilized tyrosyl radical at its active site (Lassmann etal., 1991), and nitric oxide should react directly... 

Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in biochemical transformations, once thought to be rare in biological systems. The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2 (Fig. 22-40). The R1 subunit contains two lands of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the R1 and R2 subunits. At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical. The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals (Fig. 22-40). The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis. 

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 22-41. The 3 -ribonu-cleotide radical formed in step (T) helps stabilize the cation formed at the 2 carbon after the loss of H20 (steps and (3)). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step ). Step (5) is the reverse of step ( ) regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step ). Ini , coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. 

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. 

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. 

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... 

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