The Tyrosyl Free Radical in Ribonucleotide Reductase

Abstract

The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins Bi and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of ironradical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase.The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative.In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis.Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical.

Electron Paramagnetic Resonance and Nuclear Magnetic Resonance Studies of Class I Ribonucleotide Reductase

“…A concluding remark on all free radicals hitherto observed on different amino acids in RDR is that they are formed by an oxidative process and have significant spin densities at atoms neighboring C, and, therefore, exhibit hyperfine couplings to one or both of the p protons. The CpH2 group is in a locked position relative to the sidechain at both low temperatures and at room temperature for the Y 122 radical and the azido nucleotide induced transient radical (26). This may be a general Annual Reviews www.annualreviews.org/aronline feature of a relatively stable amino acid-based free radical held in a protein environment.…”

“…of HSV or human origin. The relatively small differences in the shapes between spectra of this type from different species may be explained by small differences in the p dihedral angles, caused by the different protein environments (9,26).…”

“…This difficulty is caused by magnetic interaction with the iron center, which has a significant population of the excited paramagnetic states toward higher temperatures. Electron paramagnetic resonance spectra recorded in solution at room temperatures are broadened severely by the interaction (26,57). Electron paramagnetic resonance saturation recovery studies at variable temperature have been used to examine the interaction, which has exchange as well as dipolar components and varies significantly between proteins from different species (22).…”

Short‐Lived Radical Intermediates in the Photochemistry of Glucose Oxidase

“…On the other hand, radical forms of tyrosine (TyrOH) and tryptophan (TrpH) amino acids, that are constituents of the protein backbone, are also frequently involved as intermediates in such reactions. While in the neutral ground states both these amino acid residues absorb only in the UV region, their radical states (both protonated cation and deprotonated neutral) have distinct absorption bands in the visible region . In flavoproteins, photoreduction of excited flavin by electron transfer from nearby TyrOH or TrpH residues is an efficient fluorescence quenching mechanism.…”

Immuno-spin trapping of protein and DNA radicals: “Tagging” free radicals to locate and understand the redox process

“…The unpaired electron gives these species paramagnetic properties that make them suitable for detection by electron spin resonance (ESR) spectroscopy, the “gold standard” technique used to detect free radicals [9]. However, because of the high reactivity of protein- and DNA-centered radicals, they are generally stable for only microseconds to seconds before they decay to produce diamagnetic (ESR-silent) species; although stable protein radicals such as the tyrosyl radical of ribonucleotide reductase do exist [10]. …”