Ribonucleotide reductase tyrosyl radical cofactor

Riggs-Gelasco, P. J., Shu, L. J., Chen, S. X., Burdi, D., Huynh, B. H., Que, L., and Stubbe, J., 1998, Exafs Characterization of the intermediate X generated during the assembly of the Escherichia coli ribonucleotide reductase R2 diferric tyrosyl radical cofactor, J. Am. Chem. Soc. 120 8499860. 

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. 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

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. 

Bollinger, J. M., Tong, W. H., Ravi, N., Huynh, B. H., Edmondson, D. E., and Stuhhe, J., 1994h, Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductase III. Kinetics of the limiting Fe reaction by optical, EPR, and M ssbauer spectroscopies. /. Am. Chem. Soc. 116 8024n8032. 

Class I E. coli ribonucleotide reductase (RNR) exploits all the PCET variances of Fig. 17.3 in order to catalyze the reduction of nucleoside diphosphates to deoxynu-cleoside diphosphates. This reaction demands radical transport across two subunits and over a remarkable 35 A distance [187,188, 219]. The crystal structures of both R1 and R2 subunits have been solved independently [220-222] and a docking model has been proposed [220]. R2 harbors the diferric tyrosyl radical ( Y122) cofactor that initiates nucleotide reduction by generating a transient thiyl radical ( C439) in the enzyme active site located >35 A away in R1 [223]. Substrate conversion is initiated by a hydrogen atom abstraction (Type A PCET) at the 3 position of the substrate by C439 [192]. 

The enzyme ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deox5nibonucleotides, which is the first rate-limiting step in DNA biosynthesis. On the basis of their cofactor compositions, RNRs may be grouped into four different classes [7]. Class I RNR from E. coli is comprised of two homodi-meric subunits, R1 and R2. The R1 subunit (2 x 86kDa) contains the substrate binding site and redox-active cysteine residues, which are involved in the reduction of the ribonucleotides. The R2 subunit (2 x 43 kDa) contains in its active form (R2act) a stable tyrosyl radical (Y122 ), which is necessary for catalytic activity. This tyrosyl radical is located in close proximity to a //-oxo diferric cluster and is embedded about 10 A away from the protein surface [38, 39]. 

Bollinger Jr JM, Edmondson DE, Huynh BH, Filley J, Norton JR, Stubbe J. 1991. Mechanism of assembly of the tyrosyl radical dinuclear iron cluster cofactor of ribonucleotide reductase. Science 253 292-298. 

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. 

---