Tyrosyl radicals in ribonucleotide reductase

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. 

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. 

Denysenkov, V. P., Prisner, T. F., Stubbe, J., and Bennati, M. (2006). High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 103, 13386-13390. 

The generation, stability, and function of tyrosyl radicals in ribonucleotide reductase, PGH synthase, and galactose oxidase continue to be active areas of research. The difficulties encountered in preparing and handling these proteins, as well as in probing the physical properties and reactivity of their metal-phenoxyl radical active sites, make the preparation and investigation of stable phenoxyl radical metal model complexes an attractive goal. 

Hoganson, C. W., Sahlin, M., Sj berg, B.-M., and Babcock, G. T., 1996, Electron magnetic resonance of the tyrosyl radical in ribonucleotide reductase from Escherichia coli. J. Am. Chem. Soc. 118 467294679. 

Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM. Coimecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state. Proc. Natl. Acad. Sci. U.S.A. 2007 104 1451-1455. Denysenkov VP, Prisner TF, Stubbe J, Bennati M. High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. U.S.A. 2006 103 13386-13390. Schiemann O, Prisner TF. Long-range distance determinations in biomacromolecules by EPR spectroscopy. Quarterly Rev. Biophys. 2007 40 1-53. 

Figure 7. ESR spectra ( —196°C) of tyrosyl radicals in ribonucleotide reductase from E. coli. a, Spectrum from enzyme grown in the presence of tyrosine b, spectrum from enzyme grown in the presence of deuterated [yJ./i- HjJtyrosine. From [137], with permission.

DIFERRIC CLUSTER-TYROSYL RADICAL IN RIBONUCLEOTIDE REDUCTASE... 

Should the very simple reduction of a tyrosyl radical in ribonucleotide reductases (Eq. VI) really be the only interception of hydroxyurea with a cell s replication machinery, it has to stand up against an impressive list of consequences in eukaryotic cells. A selection of such effects, mostly observed in cell cultures, is compiled in Table 9. 

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.

Fig. 11. Numbering scheme and ENDOR-derived spin-density distributions (shown in parentheses) in the tyrosyl radical of ribonucleotide reductase. The ring carbon values are symmetry-related. Hyperfine couplings from the ring 3,5 and the /3-carbon protons are responsible for the structure of the radical EPR signal. (Adapted from Babcock and coworkers.

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-... 

Lippard et al. proposed several possible mechanisms of the alkane hydroxylation [9]. One possible mechanism is shown in Scheme 3, in which an T 2,T 2-peroxo-bridged diiron(III) acts as an active intermediate which directly transfers oxygen to an alkane substrate [9]. This mechanism suggests the participation of a mercapto radical of Cys-151. This amino acid occupies the equivalent region of space to the tyrosyl radical of ribonucleotide reductase, as indicated by sequence homology and the X-ray crystallographic results [53, 55]. 

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. 

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. 

Sahlin, M., Gr%oslund, A., Ehrenberg, A., and Sj"berg, B.-M., 1982, Structure of the tyrosyl radical in bacteriophage T4-induced ribonucleotide reductase. J. Biol. Chem. 257 366n369. 

In Ribonucleotide reductase, finally, the radical transfer mechanism between the stable tyrosyl radical in the R2 subunit and the cysteine residue at the R1 active site is outlined, and shown to primarily invoke a neutral H-atom transfer pathway, with very low barriers and thermoneutrality. In addition, the substrate mechanism is outlined, based again on a model slightly modified compared with the original experimental proposals. 

Fig. 4. Suggested long-range electron transfer pathway from the substrate binding site in protein R1 to the tyrosyl radical in protein R2 in E. coli ribonucleotide reductase. The figure is adapted from (73) with permission from B-M. Sjoberg.

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