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Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2'-Azido-2'-deoxyuridine 5'-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of -25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: gl1 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (Atz/, = 31.0 G) and upper limits (-5 G) can be placed on both A; , and A& Comparison of these g-and A-values with those of model systems in the literature suggests a structure for the radical, XN'SCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed @2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -SCH2-component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439,462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3'-keto-2'-deoxyuridine 5'-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3'-hydroxyl oxygen of the deoxyribose moiety.

The activated form of galactose oxidase from the fungus Dactylium dendroides contains a single divalent copper ion which is antiferromagnetically coupled to a protein-based free radical. Chemical oxidation of the apoenzyme generates the free radical which is localized on a covalently cross-linked tyrosine-cysteine residue. This species, together with model radicals generated by UV irradiation of protonated and selectively deuterated o-(methylthio)cresol (MTC), has been studied by high-frequency EPR spectroscopy (139.5 GHz/5 T) in conjunction with molecular orbital calculations employing self-consistent local density functional (LDF) methods. The Zeeman interactions (g values) determined from the high-frequency spectra of the apogalactose oxidase and the MTC model radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbital calculations accurately reflect the experimental data, including an increase in the axial symmetry of the Zeeman interaction for the MTC radical compared with the unsubstituted tyrosyl radical species. An explanation of this effect based on an analysis of individual atomic contributions to the molecular g values is presented. High-frequency echo-detected EPR spectroscopy of the apogalactose oxidase radical resolves hyperfine splittings. Based on the molecular orbital calculations and the EPR spectroscopic results presented here, the hyperfine splittings are assigned to two methylene protonssone derived from tyrosine and one from cysteine. These findings are consistent with the radical spin density being localized on the tyrosine-cysteine moiety, rather than delocalized throughout an extended π-network involving a nearby tryptophan as had been previously suggested as a possible explanation of the stability of the radical species.

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.