Ribonucleotide reductase tyrosyl radical

Fig. 17. High-frequency (139.5 GHz) free radical EPR spectra, (a) Oxidized apoga-lactose oxidase free radical, (b) Photochemically generated 0-(methylthio)cresyl (mtc) phenoxyl radical, (c) Ribonucleotide reductase tyrosyl radical.

Voevodskaya N, Narvaez AJ, Domkin V, Torrents E, Thelander L, Graslund A. 2006. Chlamydial ribonucleotide reductase tyrosyl radical function in catalysis replaced by the Fe We cluster. Proc Nad Acad Sci USA 103 9850-9854. 

Ribonucleotide reductase is responsible for the conversion of the four biological ribonucleotides (RNA) into their corresponding deoxy forms (DNA). Although RNR is not an oxygenase during its primary catalyzed reaction (the conversion of ribonucleotides), it activates oxygen to generate a stable tyrosyl radical that is essential to the overall mechanism [46 49]. The common link between the chemistry of MMO and RNR is the activation of O2 by the di-iron active site. 

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. 

M. Bennati, A. Weber, J. Antonie, D.L. Perlstein, J. Robblee and J. Stubbe, Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl radicals in the R2 subunit of the E. coli ribonucleotide reductase, J. Am. Chem. Soc., 2003, 125, 14988. 

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. 

One of the most powerful spectroscopic techniques for the detection and characterization of persistent and transient phenoxyls is time-resolved resonance Raman (RR) spectroscopy. Vibrational frequencies and the relative intensities of the resonance-enhanced bands have proven to be sensitive markers for tyrosyl radicals in proteins. For example, Sanders-Loehr and co-workers (31) detected the tyrosyl radical in native ribonucleotide reductase from Escherichia coli by a resonance-enhanced Raman mode at 1498 cm 1 that they assigned to the Ula Wilson mode of the tyrosyl, which is predominantly the u(C=0) stretching mode. 

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... 

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.

A four-pulse DEER measurement of the distance between two tyrosyl radicals on the monomers that make up the R2 subunit of E. coli ribonucleotide reductase gave a point-dipole distance of 33.1 A, which is in good agreement with the X-ray crystal structure.84 Better agreement between the calculated and observed dipolar frequency could be obtained by summing contributions from distributed... 

In subunit R2 of ribonucleotide reductase there is a tyrosyl radical (Y ) in close proximity to a di-iron cluster.100 In the protein from E. coli the EPR signal from Y can be observed up to room temperature. However, in the protein from yeast the Y signal broadens above 15 K and is not observable above about 60 K. Saturation recovery measurements at 140 GHz showed that at 60 K the spin-lattice relaxation rates for the Y signal in the yeast protein were about 2 orders of magnitude faster than for the E. coli protein. The temperature dependence of the relaxation enhancement was consistent with the activation energy for the first excited state of the di-iron cluster, so the relaxation enhancement was attributed to interaction with the di-iron cluster. Relaxation enhancements measured at 140 GHz showed little orientation dependence so the enhancement was assigned to isotropic exchange, which is different from the orientation-dependent dipolar interaction observed for the E. coli protein.100... 

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. 

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. 

A chain of hydrogen-bonded side chains apparently provides a pathway for transfer of an impaired electron from the active site to the Tyr 122 radical and from there to the radical generating center.363 The tyrosyl radical can be destroyed by removal of the iron by exposure to 02 or by treatment of ribonucleotide reductases with hydroxyurea, which reduces the radical and also destroys catalytic activity ... 

Three types of ribonucleotide reductase catalyze this reduction of the ribose ring. The most widely distributed in nature occurs in mammalian and plant cells, in yeast, and in some prokaryotes. This type of reductase contains a tyrosyl radical closely associated with nonheme iron, as in the reductase from E. coli. The E. coli reductase is composed of two nonidentical subunits, both contributing to the active site it is specific for the reduction of diphosphates (ADP, GDP, CDP, and UDP). 

An unusual feature of ribonucleotide reductase is that the reaction it catalyzes involves a radical mechanism. The mammalian type of reductase initiates this reaction by the tyrosyl radical-nonheme iron. Hydroxyurea and related inhibit the majTrrrraiVarr retftrcCase 6y abolishing the radical state of the tyrosine residue. Inhibition of DNA synthesis by such compounds is secondary to this effect. 

Hydroxyurea interferes with the synthesis of both pyrimidine and purine nucleotides (see table 23.3). It interferes with the synthesis of deoxyribonucleotides by inhibiting ribonucleotide reductase of mammalian cells, an enzyme that is crucial and probably rate-limiting in the biosynthesis of DNA. It probably acts by disrupting the iron-tyrosyl radical structure at the active site of the reductase. Hydroxyurea is in clinical use as an anticancer agent. 

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