An ENDOR study of the tyrosyl free radical in ribonucleotide reductase from Escherichia coli

Abstract

Tyrosyl radicals have been identified as components of several proteins whose function is redox chemistry. Ribonucleotide reductase is representative of this class of enzymes as its B2 subunit contains a tyrosine residue that is necessarily a radical for activity. ENDOR spectroscopy is a higher resolution technique, and we used it to characterize the enzyme-bound radical in detail. ENDOR enhancement in ribonucleotide reductase is observed only at temperatures below 110 K due to a temperature-dependent relaxation enhancement of the radical by a μ-oxo-bridged pair of high-spin ferric ions. Below this temperature, excellent spectra are obtained and specific deuteration of the tyrosine residues in the protein was used to assign measured hyperfine tensors to the various protons in the radical. An analysis of the principal tensor components of the ortho protons (A_x = −26.9 MHz, A_y= −7.8 MHz, A_z = −19.7 MHz) and the strongly coupled β-methylene proton establishes that the radical has characteristics of a seven-member odd-alternate species with a spin density distribution of 0.16 (phenol oxygen), 0.26 (ortho), −0.07 (meta), −0.03 (ring carbon carrying phenol oxygen), and 0.49 (para). These calculations also provide a determnation of the McConnell Q value for ring protons in this class of radical. The β protons are situated with dihedral angles of 30° and 90° with respect to the P_I orbital on carbon-1 of the aromatic ring. The results indicate that the tyrosyl radical of ribonucleotide reductase is uncharged and not hydrogen bonded to donors in its environment within the protein.