ELECTRON MAGNETIC-RESONANCE OF THE TYROSYL RADICAL IN RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI

The spin density distribution of the Y-122 tyrosyl radical in the R2 s ubunit of ribonucleotide reductase from Escherichia coli has been dete rmined. Incorporation of isotopically labeled tyrosine into the protei n has allowed us to measure the O-17 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 dete rmination of the orientation of the hyperfine tensors relative to the g tenser. Electron-nuclear-nuclear triple resonance has been applied t o establish the relative signs of three hyperfine couplings. These mea surements give a more precise and more accurate picture of the spin de nsity distribution in a protein tyrosyl radical than has been availabl e previously. The O-17 hyperfine splitting in tyrosyl radicals in aque ous glasses has also been measured. The differences in hyperfine coupl ings indicate that addition of a hydrogen bond to the phenolic oxygen perturbs the spin density in the ring slightly and causes the spin den sity at the oxygen atom to decrease by about 10%. Comparison of our re sults for the ribonucleotide reductase Y-122 tyrosyl radical with thos e for other naturally occurring tyrosyl radicals and with tyrosines in aqueous glasses shows that there is only slight variation in spin den sity distribution over the phenol ring in this class of radicals, desp ite substantial variation in local environment.