Sensitivity of tyrosyl radical g-values to changes in protein structure: Ahigh-field EPR study of mutants of ribonucleotide reductase

The local electrostatic environment plays a critical role in determining th e physicochemical properties of reactive radicals in proteins. High-field e lectron paramagnetic resonance (HF-EPR) spectroscopy has been used to deter mine the sensitivity of the tyrosyl radical g-values to local electrostatic environment. Site-specific mutants of ribonucleotide reductase from Escher ichia coil were used to study the effect of introducing a charge group on t he HF-EPR spectrum of the stable tyrosyl (Y122) radical. The changes affect ed by the mutations were small, but measurable. Mutation of isoleucine-74 t o an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine int eractions. Similar effects were observed for the mutation of valine-136 to an arginine (V136R) or asparagine (V136N). For five or six mutants studied, the g(x) component of the g-tensor was distributed. For the isoleucine-74 to lysine (I74K) and leucine-77 to phenylalanine (L77F) mutants, a shift of 1 x 10(-4) in g(x) value was also detected. For the I74K mutant, it is sho wn that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of th e nearby diiron center. For the L77F mutant, the shift is induced by the di iron center. Using existing tyrosyl radical g-tensor measurements, we have developed a simple effective charge model that allows us to rationalize the effect of the local electrostatic environments in a number of proteins.