Free radicals in ribonucleotide reductases

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. 

Atkin, C. L., Thelander, L., Reichard, P., and Lang, G., 1973, Iron and free radical in ribonucleotide reductase. Exchange of iron and M sshauer spectroscopy of the protein B2 subunit of the Escherichia coli enzyme. J. Biol. Chem. 248 7464n7472. 

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 ... 

Barlow T, Eliasson R, Platz A, Reichard P, Sjdberg B-M. 1983. Enzymic modification of a t5Tosine residue to a stable free radical in ribonucleotide reductase. Proc Natl Acad Sci USA 80 1492-1495. 

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. 

Lepoivre, M., Flaman, J., Bobe, P., Lemaire, G., and Henry, Y. (1994). Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide Relationship in cytostasis induced in tumor cells by cytotoxic macrophages.. Biol. Chem. 269, 21891-21897. 

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.)...

Studies on three different iron-sulfur enzyme systems, which all require S-adenosyl methionine—lysine 2,3-aminomutase, pyruvate formate lyase and anaerobic ribonucleotide reductase—have led to the identification of SAM as a major source of free radicals in living cells. As in the dehydratases, these systems have a [4Fe-4S] centre chelated by only three cysteines with one accessible coordination site. The cluster is active only in the reduced... 

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... 

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. 

FIGURE 6. The role of free radicals in the reduction of ribonucleotides to 2 -deoxyribonu-cleotides catalyzed by AdoCbl-dependent ribonucleotide triphosphate reductase. 

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

Persson, A. L., Salin, M., and Sj berg, B.-M., 1999, Transient free radicals in the reaction witii mutant E441Q Rl, wild-type R2 and CDP of Escherichia coli class la ribonucleotide reductase. Manuscript in preparation. 

Sahlin, M., Lassmann, G., Putsch, S., Sj berg, B.-M., and Gr%oslund, A., 1995, Transient free radicals in iron/oxygen reconstitution of mutant protein R2 Y122F6Possible participants in electron transfer chains in ribonucleotide reductase. J. Biol. Chem. 270 12361nl2372. 

Figure 4 a) X-band EPR spectra of tyrosyl free radical in (i) E. coli, (ii) Mycobacterium tuberculosis, and (iii) mouse ribonucleotide reductase R2 proteins (1 7). All spectra were obtained under nonsaturation conditions at 20 K. b) Spin density distribution of the unpaired electron obtained from Isotope-labeling EPR studies, c) The distances between the phenolic oxygen of tyrosyl radical and the nearest Fe ion deduced from the relaxation properties of the tyrosyl radicals. 

Hydroxyurea, an antineoplastic agent, acts by destroying an essential free radical in the active center of ribonucleotide reductase. 

Hydroxyurea is an inhibitor of the enzyme ribonucleotide reductase. Hydroxurea s mechnism of inhibition involves destruction of the free radical in the enzyme that is essential for its activity (Figure 21.15). 

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 ribonucleotide reductases (RNRs) catalyze the deoxygenation of nucleotides in the ratedetermining step of the biosynthesis of DNA. These enzymes have been categorized into three classes, each of which incorporates a metal site that initiates nucleotide reduction via processes that involve free radicals, in particular cysteinyl radicals. " Extensive studies of these processes have provided detailed insights into their mechanisms, as described in comprehensive reviews. Here we focus on the most thoroughly characterized class I enzymes, 4oi which in their resting state contain a stable tyrosyl radical in close proximity, but not directly coordinated, to a diiron cluster. 

The g-selection effect is also observed in the case of free radicals for which there is g-anisolropy. For example, the H-ENDOR hyperfine spectra of the tyrosyl radical of ribonucleotide reductase exhibits dramatic selectivity by the g-selection technique. Figure 3 depicts the selectivity obtained near die so-called matrix region of the ENDOR spectrum. At g= 1.99 the matrix region, which corresponds to pro-... 

Graslund A. 2002. Ribonucleotide reductase Kinetic methods for demonstrating radical transfer pathway in protein R2 of mouse enzyme in generation of tyrosyl free radical. In Enzyme kinetics and mechanism, Pt F detection and characterization of enzyme reaction intermediates, pp. 399 14. New York Academic Press. 

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