Escherichia coli, ribonucleotide reductase

Davydov, R., Kuprin, S. Graslund, A., and Ehrenberg, A. 1994. Electron paramagnetic resonance study of the mixed-valent diiron center in Escherichia coli ribonucleotide reductase produced by reduction of radical-free protein R2 at 77 K. Journal of the American Chemical Society 116 11120-11128. 

Cyclic voltammetry has been also used for estimation of the rate constants for oxidation of water-soluble ferrocenes in the presence of HRP (131). There is a perfect match between the data obtained spectrophotometrically and electrochemically (Table IV), which proves that the cyclic voltammetry reveals information on the oxidation of ferrocenes by Compound II. It is interesting to note that an enzyme similar to HRP, viz. cytochrome c peroxidase, which catalyzes the reduction of H202 to water using two equivalents of ferrocytochrome c (133-136), is ca. 100 times more reactive than HRP (131,137). The second-order rate constant equals 1.4 x 106 M-1 s 1 for HOOCFc at pH 6.5 (131). There is no such rate difference in oxidation of [Fe(CN)e]4- by cytochrome c peroxidase and HRP (8). These comparisons should not however create an impression that the enzymatic oxidation of ferrocenes is always fast. The active-R2 subunit of Escherichia coli ribonucleotide reductase, which has dinuclear nonheme iron center in the active site, oxidizes ferrocene carboxylic acid and other water-soluble ferrocenes with a rate constant of... 

Nordlund, P., and Eklund, H., 1993, Structure and function of the Escherichia coli ribonucleotide reductase protein R2 J. Mol. Biol. 232 1239164. 

Dong, Y., Kauffmann, K., M,nck, E., and Que, L., Jr., 1995a, An exchange-coupled complex with localized high-spin Fe and Fe sites of relevance to cluster X of Escherichia coli ribonucleotide reductase, J. Am. Chem. Soc. 117 11377nll378. 

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. 

Climent, L, Sj berg, B.-M., and Huang, C. Y., 1991, Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction Kinetic analysis of inhibition studies. Biochemistry 30 5164n5171. 

Karlsson, M., Sahlin, M., and Sj"berg, B.-M., 1992, Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme. 

Lawrence, C. C., Bennati, M., Obias, H. V., Bar, G., Griffin, R. G., and Stubbe, J., 1999, High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5 -diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 96 8979ii8984. 

Parkin, S. E., Chen, S. X., Ley, B. A., Mangravite, L., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., 1998, Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the E208Y mutant of Escherichia coli ribonucleotide reductase protein R2. Biochemistry 37 112491130. 

One example of an enzyme-catalyzed reaction involving a radical intermediate is the enzyme ribonucleotide reductase, which catalyzes the conversion of ribonucleotides (used for RNA biosynthesis) to 2 -deoxyribonucleotides (used for DNA biosynthesis), as illustrated in Fig. 16. Spectroscopic studies of the R2 subunit of Escherichia coli ribonucleotide reductase have shown that it can form a stable, long-lived, tyrosyl radical species—the first protein radical to be discovered (13). 

Seyedsayamdost, M. R., Yee, C. S., Reece, S. Y., et al. (2006) pH rate profiles of FnY356-R2s (n = 2, 3,4) in Escherichia coli ribonucleotide reductase Evidence that Y-356 is a redox-active amino acid along the radical propagation pathway. Journal of the American Chemical Society, 128(5), 1562-1568. 

A specific example is the combined multifrequency EPR and 35 GHz ENDOR study of the dLiron center in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) Samples in natural isotopic abundance (0.038% 0) and using H2 0 (34.9% 0) or 02 (85.5% 0) were prepared. ENDOR provided 0 (/ = 5/2) hyperfine coupling constants and these values were used to quantify the line broadening seen in 0-enriched versus natural abundance samples, which showed that the intermediate X contained two oxygen atoms, initially derived from dioxygen, as opposed to an alternate model with only one such oxygen atom. 

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.

Krebs C, Chen S, Baldwin J, Ley BA, Patel U, Edmondson DE, Huynh BH, Bollinger Jr MJ. 2000. Mechanism of rapid electron transfer during oxygen activation in the r2 subunit of Escherichia coli ribonucleotide reductase, 2 evidence for and consequences of blocked electron transfer in the W48F vansaA.JAm Chem Soc 122 12207-12219. 

Gerez C, Fontecave M. 1992. Reduction of the small subunit of Escherichia coli ribonucleotide reductase by hydrazines and hydroxylamines. Biochemistry 31 780-786. 

Ravi N, Bollinger Jr JM, Huynh BH, Edmondson DE, Stubbe J. 1994. Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of Escherichia coli ribonucleotide reductase, 1 Mossbauer characterization of the diferric radical precursor. J Am Chem Soc 116 8007-8014. 

Tong W, Burdi D, Riggs-Gelasco P, Chen S, Edmondson D, Huynh BH, Stubbe J, Han S, Arvai A, Tainer J. 1998. Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography. Biochemistry 37 5840-5848. 

Burdi D, Sturgeon BE, Tong WH, Stubbe JA, Hoffman BM. 1996. Rapid freeze-quench ENDOR of the radical X intermediate of Escherichia coli ribonucleotide reductase using 02, H2 0, and H20. JAm Chem Soc 118 281—282. 

Bollinger Jr JM, Chen SX, Parkin SE, Mangravite LM, Ley BA, Edmondson DE, Huynh BH. 1997. Differential iron(II) affinity of the sites of the diiron cluster in protein R2 of Escherichia coli ribonucleotide reductase tracking the individual sites through the O2 activation sequence. JAm Chem Soc 119 5976-5977. 

Bollinger Jr JM, Krebs C, Vicol A, Chen SX, Ley BA, Edmondson DE, Hnynh BH. 1998. Engineering the diiron site of Escherichia coli ribonucleotide reductase protein R2 to accumulate an intermediate similar to Hpercnio> the putative peroxodhron(III) complex from the methane monooxygenase catalytic cycle. J Am Chem Soc 120 1094-1095. 

Lu S, Lihhy E, Saleh L, Xing G, Bollinger Jr JM, Moeime-Loccoz P. 2004. Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants implications for the O2 activation mechanism. J Biol Inorg Chem 9 818-827. 

Liu A, Sahlin M, Potsch S, Sjdberg BM, Graslund A. 1998. New paramagnetic species formed at the expense of the transient tyrosyl radical in mntant protein R2 F208Y of Escherichia coli ribonucleotide reductase. Biochem Biophys Res Commun 246 740-745. 

Kolberg M, Bleifuss G, Sjdberg B-M, Graslund A, Lubitz W, Lendzian F, Lassmann G. 2002. Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase. Arch Biochem Biophys 397 57-68. 

Persson AL, Eriksson M, Katterle B, Pdtsch S, Sahlin M, Sjdberg B-M 1997. A new mechanism-based radical intermediate in a mutant R1 protein affecting the catalyti-caUy essential Glu(441) in Escherichia coli ribonucleotide reductase. J Biol Chem 272 31533-31541. 

---