Ribonucleotide reductase B2 subunit

Figure 3. First shell (1.1-2.3A) EXAFS spectra of methemerythrin azide, semimethemerythrin azide, ribonucleotide reductase B2 subunit, oxidized uteroferrin-phosphate complex, and reduced uteroferrin.

Ribonucleotide reductase ribonucleoside diphosphate reductase) is a multisubunit enzyme (two identical B1 subunits and two identical B2 subunits) that is specific for the reduction of nucleoside diphosphates (ADP, GDP, CDP, and UDP) to their deoxy-forms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2-hydroxyl group are two sulfhydryl groups on the enzyme itself, which, during the reaction, form a disulfide bond (Figure 22.12). 

Fig. 9. Resonance Raman spectrum of tyrosine radical in purified E. coli ribonucleotide reductase (A) Pure B2 subunit (B) holoenzyme (C) metB2 subunit (lacking tyrosine radical). Note the correlation of the 1498 cm-1 band with the presence of the tyrosine radical. Reprinted with permission from Backes, G., Sahlin, M., Sjoberg, B.-M., Loehr, T.M. and Sanders-Loehr, J. (1989) Biochemistry 28, 1923-1929. Copyright 1989, American Chemical Society.

The metabolic function of the thioredoxin reductase-thioredoxin system is to supply reducing equivalents to a wide variety of acceptors. By far the best characterized of these is the E. coli ribonucleotide reductase system (23, 261) the reductase consists of two subunits, proteins B1 and B2 (262, 263), The B1 protein contains three reactive dithiol-disulfide pairs and appears to be the immediate acceptor of reducing equivalents from thioredoxin. As isolated, the three pairs are in the reduced state and, in the presence of the B2 protein, three molecules of ribonucleotide can be reduced prior to any input of reducing equivalents from thiore-... 

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. 

Ribonucleotide reductase of E. coli consists of two non-identical subunits, which are purified together during the initial steps of the purification procedure however upon further purification the reductase separates into two subunits, proteins B1 and B2 (58). The overall yield of the two proteins is low, in particular that of protein Bl, which readily decomposes into smaller subunits. This decomposition can be counteracted by the addition of dithiols. An improved procedure for the purification of protein B1 using affinity chromatography on dATP-Sepharose has been described by Thelander (59). 

When protein B1 and B2 are mixed in the presence of magnesium ions and dithiothreitol active ribonucleotide reductase with an S2o,w of 9.7S is formed. Stimulatory effectors, such as ATP and TTP, do not effect complex formation. In contrast, in the presence of the negative effector, dATP, at concentrations which inhibit enzyme activity a larger complex is formed with an S20, w of 15.5S. Both complexes contain equimolar amounts of each subunit. A heavy complex is also formed in the presence of mixtures of other nucleoside triphosphates which inhibit enzyme activity. On the other hand the formation of this heavy inactive complex is prevented by ATP at concentrations which reverse the inhibition by dATP (63). More recent experiments (59) have shown that the interaction between proteins B1 and B2 in the presence of dATP is strongly influenced by the presence of sucrose, and indeed in the absence of sucrose subunits B1 and B2 with dATP form a complex with an S20, w of 22.1S. 

All non-heme iron containing ribonucleotide reductases are also inhibited by hydroxyurea and related hydroxamates, while the adenosyl-cobalamin-dependent reductases are not affected (27, 156). The inhibition by these reagents can be partially reversed by excess Fe+2 or dithiols. Reaction of ribonucleotide reductase of E. coli with [14C]hydro-xyurea inactivated only the B2 subunit and this inactivation was not reversed by removal of the radioactivity (157). Inactivation by hydroxyurea does not affect the iron content of protein B2, but involves the destruction of the stable free radical (66,67). Reactivation can be accomplished by removal of the iron and reconstitution of apoprotein B2 with Fe+2. Hydroxyurea has been demonstrated to be a powerful radical scavenger in another system (158). 

From their key role in DNA synthesis it is not surprising that the ribonucleotide reductases are ubiquitous in nature. However, three different types of ribonucleotide reductases are now known, each with variations in their cofactor requirements. The most extensively studied and characterized are the ribonucleotide-diphosphate reductases (RDPRs), in particular the enzyme from Escherichia coli. This reductase consists of two nonidentical subunits, proteins B1 and B2, which form an active 1 1 complex where the interface between these subunits forms the active site, and each subunit alone is devoid of catalytic activity (9). Protein B2 (Mr 78,(XK)) is a dimer and contains two atoms of tightly... 

The FeRR system has been extensively studied (112, 114). For this type of ribonucleotide reductase, the enzyme is comprised of two subunits, known as R1 and R2 (or occasionally B1 and B2). The R1 and... 

Figure 3. (a) Representation of the structure of the B2 subunit of the E. coli ribonucleotide reductase. Courtesy of Prof. Hans Eklund. b) Schematic of the binuclear iron site in the B2 subunit of ribonucleotide reductase showing the dissimilar iron sites in the x-oxo bridged core. The relative position of the tyr,22 radical that is 5 A from the diiron site is indicated. 

Recently, a new type of ribonucleotide reductase that did not require coenzyme-B12 or iron for activity was discovered in the coryneform bacteria Brevibacteriium ammoniagenes and Micrococcus luteus (228) this enzyme requires manganese instead. Like the diiron-containing enzyme from E. coli, the enzyme from B. ammoniagenes consists of two components, a 30 kDa B1 subunit that binds the nucleotides and 100 kDa B2 subunit consisting of two 50-kDa chains with at least one Mn per chain... 

Enzyme from Mn-deficient cells showed no ribonucleotide reductase activity but could be activated by addition of Mn (215). Furthermore, Mn was incorporated into the B2 subunit when the bacteria were grown on MnCL-enriched medium (8). These experiments strongly implicate a manganese containing active site. 

The electronic spectrum of the B2 subunit ( ax = 455, 485, and 615 nm) closely resembles those of Mn-catalase and synthetic tribridged Mn 0 complexes (8). The metal site was thus proposed (229) to be analogous to the diiron center of the enzyme from E. coli. This analogy may be reasonable as iron restores 50-70% of the activity in protein derived from Mn-deprived cells (230). Similar to the enzyme from E. coli, the Mn-containing ribonucleotide reductase is inhibited by hydroxyurea and au-... 

Figure J (a) (Que and True). Representation of the structure of the B2 subunit of the E. colt ribonucleotide reductase. 

Fig. 2. EPR spectra of tyrosine radicals (A) the stable tyrosine radical, Tyr[,-, in photosystem II (65 ij,M) in a frozen solution containing 30% (v/v) ethylene glycol in water and (B) the stable tyrosine radical in the B2 subunit of Escherichia coli ribonucleotide reductase (50 jtiAf) in a frozen solution containing 10% (v/v) ethylene glycol in water. Conditions temperature, 8.0 K microwave frequency, 9.05 GHz magnetic field modulation amplitude, 2 G magnetic field modulation frequency, 100 kHz microwave power, 0.7 /iW.

Fig. 5. Progressive microwave power saturation plot of the tyrosine radical in the B2 subunit of E. coli ribonucleotide reductase. Conditions were as in Fig. 2B.

The effects of hydroxyurea in the purified ribonucleotide reductase systems of E. coU, phage T4, calf thymus and mouse cells have been described above (p. 36, 42). Inhibition of substrate reduction in vitro (I50 = 2 - 3 10 ) is accompanied by loss of the tyrosyl radical, but not iron from E. coli subunit B2. Studies with substituted hydroxyl-amines and hydroxamates showed good correlation between their ability to undergo one-electron oxidation and enzyme inhibition, unless branched substituents prevented interaction with the protein (Table 8) Thus the mode of inhibition of E. coli ribonucleotide reductase is essentially solved Within steric restrictions of accessibility to the active site the compounds donate an electron to the enzyme s free radical, producing an inactive protein with still intact binuclear iron complex (Eq. VI). This process is irreversible in vitro until iron is removed, and then reintroduced with Fe(II)ascorbate in the presence of oxygen, whereupon radical and enzyme activity reappear. No other enzyme of E. coli has been found to be inactivated by hydroxyurea. 

As purification of the ribonucleotide reductase progressed, it became evident that the activity was present in a complex of two protein components, B1 and B2 these are now known to be nonidentical subunits of a remarkable allosteric enzyme. It was recognized that dihydrolipoate was unlikely to be the physiological reductant in this system. 

These results have been explained by a mechanism which supposes that a hydride ion (H ) is generated and that a concerted reaction takes place in which the leaving group (OH ) dissociates from C-2, but only when the attacking species (H ) is present (7, W). Beichard has sug sted that the iron-containing B2 subunit of the E. colt ribonucleotide reductase may somehow provide the postulated hydride ion. In ribonucleotide reduction by the L. leichmannii system, 5 -deoxyadenosylcobalamin participates as a coenzyme and may also be involved in generating a hydride ion. 

Ribonucleotide reductase of Escherichia coli, which catalyzes the reduction of ribonucleoside 5 -diphosphates to 2 -deoxynucleoside 5 -diphos-phates, consists of two nonidentical subunits, proteins B1 and B2. In the presence of Mg , the two subunits form a 1 1 complex of active enzyme. When separated, neither subunit has any known biological activity. Protein B1 has a molecular weight of 160,000, contains the active dithiols, is capable of interacting with thioredoxin, and contains... 

---