Ribonucleotide reductase oxygen activation

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

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. 

The function of the metal site in the oxygen-dependent radical enzymes galactose oxidase, amine oxidases, ribonucleotide reductase, and cytochrome c oxidase is inter alia to bind 02 in their reduced forms and undergo the appropriate redox chemistry to generate a metal-bound, activated oxygen species of variable nature. 

Fig. 14. Intermediates in the activation of oxygen by binuclear iron centers. Hr, Heme-rythrin RNR, ribonucleotide reductase MMO, methane monooxygenase.

Que L Jr, Dong YH. Modeling the oxygen activation chemistry of methane monooxygenase and ribonucleotide reductase. Acc Chem Res. 1996 29 190-6. 

Whilst more is still to be learnt about the growth-promoting effect of active oxygen species, there is of course the quite specific radical involvement in the activity of the enzyme ribonucleotide reductase [143] which catalyses the reduction of ribonucleotides to deoxyribonucleotides, an essential step in the DNA-synthetic S-phase of the cell cycle. 

Fig. 9. Redox-active amino acid residues related to tyrosine, (a) Tyrosine, the redox center in ribonucleotide reductase, prostaglandin H synthase, and the photosynthetic oxygen evolving complex, (b) 2,4,5-Trihydroxyphenylalanine, the redox cofactor of the quinoprotein amine oxidase, (c) Tyrosine-cysteine (Tyr-Cys), the redox cofactor of galactose oxidase.

Andersson, M. E., H"gbom, M., Rinaldo-Matthis, A., Andersson, K. K., Sj berg, B.-M., and Nordlund, P., 1999, The crystal structnre of an azide complex of the diferrous R2 subunit of ribonucleotide reductase displays a novel carboxylate shift with important mechanistic implications for diiron-catalyzed oxygen activation. J. Amer. Chem. Soc. 121 2346n2352. 

Elgren, T. E., Lynch, J. B., Juarez-Garcia, C., M,nck, E., Sj berg, B.-M., and Que, L. J., 1991, Electron transfer associated with oxygen activation in the B2 protein of ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 266 19265nl9268. 

Logan, D. T., Su, X. D., berg, A., Regnstr m, K., Hajdu, J., Eklund, H., and Nordlund, P., 1996, Crystal structure of reduced protein R2 of ribonucleotide reductase The structural basis for oxygen activation at a dinuclear iron site. Structure 4 1053nl064. 

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. 

The role of oxygen in eukaryotic DNA biosynthesis may indeed be a critical one. It has recently been shown that O2 is not only required for initial formation of tyrosyl radical but must be continously present to maintain the radical content and enzyme activity of mammalian ribonucleotide reductase In vivo studies with Ehrlich ascites cells also point to a tight link between oxygen and deoxyribonucleotide supply Anaerobic arrest of cells in G1 phase and block of DNA synthesis can be relieved by addition of deoxycytidine, but not cytidine, to the culture medium. ... 

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. 

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