No abstract

Two different tryptophan radicals (Wa • and Wb •) with lifetimes of several minutes at room temperature are formed during the reconstitution of the diiron center in the Escherichia coli ribonucleotide reductase mutant protein R2 Y122F. Detailed hyperfine parameters are for the first time determined for protein-linked oxidized neutral tryptophan radicals. Wa • is freeze-trapped and investigated by EPR and ENDOR in protonated and selectively deuterated proteins at 20 K. Two hyperfine couplings from the β-methylene protons, hyperfine tensors of two α-protons, and the complete nitrogen hyperfine tensor are determined. Based on the absence of a large hyperfine coupling from the N−H proton, which would be expected for a cation radical, and on comparison of the experimental data with theoretical spin densities from density functional calculations, Wa • is assigned to an oxidized neutral tryptophan radical. A small anisotropic hyperfine coupling detected in selectively deuterated Wa • is tentatively assigned to a proton which is hydrogen bonded to the nitrogen of Wa •. A similar spin density distribution as for Wa • is obtained also for the second tryptophan radical, Wb •, observed by EPR at room temperature, which is also assigned to an oxidized neutral radical.

The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by sitedirected mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2 -azido-2 -deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDPderived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogenbonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.The enzyme ribonucleotide reductase is essential for all living organisms. By catalyzing the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the enzyme furnishes cells with precursors for DNA synthesis. To maintain a stable and balanced supply of nucleotides during cell proliferation, the enzyme is cell cycle regulated (1) and also under strict allosteric regulation (for recent review see Ref.2). The Escherichia coli ribonucleotide reductase holoenzyme complex consists of two nonidentical dimeric proteins, R1 and R2, which are each inactive alone. The larger protein R1 contains substrate and allosteric effector binding sites. The substrate binding site of R1 includes a cysteine triad that is involved in catalysis (3-5). The smaller protein R2 contains an essential tyrosyl radical that is generated and stabilized by an oxo-bridged diiron center (6 -9). The oxidized tyrosyl radical of R2 probably participates in the reaction mechanism as a transient electron sink.The reaction catalyzed by ribonucleotide reductase is the reduction of the 2Ј-hydroxyl group of a ribonucleoside diphosphate. The mechanism involves the initial generation of a transient protein radical in R1 close to the bound substrate. The protein radical abstracts a hydrogen atom from the 3Ј position of the substrate thereby generating an oxidized substrate radical that enables leaving the protonated 2Ј-hydroxyl group. The resulting substrate radical cation intermediate is reduced by a redox-active cysteine pair, which in turn is oxidized to a disulfide (10). The 3Ј-hydrogen atom is reintroduced by the same amino acid that initially abstracted it and that again forms the transient protein radical (11).The crystal structure of the R2 protein shows that the R2 radical is buried inside the protein structure about 10 Å from the closest s...

The spin density distribution of the Y 122 tyrosyl radical in the R2 subunit of ribonucleotide reductase from Escherichia coli has been determined. Incorporation of isotopically labeled tyrosine into the protein has allowed us to measure the 17 O hyperfine coupling by using EPR, giving a direct measure of the tyrosine phenol oxygen spin density, 0.29 ( 0.02. The hyperfine tensors of six protons of the radical have been determined by using ENDOR. Magnetic field selection allows a determination of the orientation of the hyperfine tensors relative to the g tensor. Electron-nuclear-nuclear triple resonance has been applied to establish the relative signs of three hyperfine couplings. These measurements give a more precise and more accurate picture of the spin density distribution in a protein tyrosyl radical than has been available previously. The 17 O hyperfine splitting in tyrosyl radicals in aqueous glasses has also been measured. The differences in hyperfine couplings indicate that addition of a hydrogen bond to the phenolic oxygen perturbs the spin density in the ring slightly and causes the spin density at the oxygen atom to decrease by about 10%. Comparison of our results for the ribonucleotide reductase Y 122 tyrosyl radical with those for other naturally occurring tyrosyl radicals and with tyrosines in aqueous glasses shows that there is only slight variation in spin density distribution over the phenol ring in this class of radicals, despite substantial variation in local environment.