The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122 • ). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-Å x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122 • obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography. R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides and are thus essential for DNA synthesis. Class I RNRs consist of two homodimeric proteins: R1, which contains the active site and binding site for allosteric regulators, and R2, which generates and harbors the tyrosyl radical Y122• (Escherichia coli numbering) needed for catalysis. The catalytic reaction in R1 is believed to be initiated by a reversible radical transfer from Tyr 122 in R2 to an active-site cysteine in R1.The tyrosyl radical on the R2 subunit is generated by means of the reductive cleavage of molecular oxygen at a diiron center. Crystal structures of E. coli R2 are available for both the reduced diferrous (Fe 2). The structural data have, together with kinetic data and theoretical calculations, served as the basis for the formulation of proposals for the mechanism of radical generation and radical migration in the overall RNR reaction (3-9). However, no structure has been available for the radical-containing form, hence, the orientation and location of the active radical Y122• have not been known. The diiron center is in the active enzyme in the diferric form and couples antiferromagnetically to an S ϭ 0 ground state (3-5, 10). Detailed high-field EPR experiments have been performed on Y122• in frozen R2 solutions (11-15). The obtained g-tensor values were found to be indicators for the polarity of the radical environment. In particular, it was found that the tyrosyl radical is hydrogen-bonded in RNR of mouse and herpes simplex virus (12, 14), whereas in E. coli it is not (11-15).In...

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.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.

Structural alterations of albumin, their dependence on concentration and the role of free -SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to p structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of b structures and aggregates increases.At degrees of denaturation of up to 20';;; complete renaturation is possible in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional a-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free -SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethylmaleimide to free -SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65 -70 "C, oligomers are formed mainly via intermolecular p structures.In the preparation of human serum albumin for clinical purposes its behaviour at different temperatures is of importance. The albumin is treated at 60 "C for about 10 h to inactivate the hepatitis virus. The structure should be largely retained of course in this treatment. Sodium octanoate or sodium octanoate + acetyltryptophan may be used as stabilizer. We distinguish in general two stages in the heat treatment of albumin. The first stage includes reversible structural alterations, the second one includes irreversible structural alterations, which may not necessarily result in a complete destruction of the ordered structure [l--31. Although a number of investigations are available on the problem of the thermal exposure of albumin, there are still open questions to be answered concerning in particular the nature of structural alterations, the limits of reversibility, the influence of concentration and environmental conditions and the molecular mechanism of the action of the stabilizers and their relative effectiveness under various conditions. Experiments perAbbreviations. H + 'H exchange, hydrogen -deuterium exchange; ESR, electron spin resonance; CD, circular dichroism; MalNEt, N-ethylmaleimide.formed on horse serum albumin by Zimmermann and Dittmar [4] showed that higher molecular weight components will be increasingly formed after heat treatment of 15 min at 100 "C. If the time of exposure is 60min or more, the aggregation products will decompose into low-molecular-weight fragments which are serologically inactive. They may still cause, however, severe shock reactions in anaphylaxia experiments. The authors suggest that species des...