PMID:11457015
| Citation |
Un, S, Gerez, C, Elleingand, E and Fontecave, M (2001) Sensitivity of tyrosyl radical g-values to changes in protein structure: a high-field EPR study of mutants of ribonucleotide reductase. J. Am. Chem. Soc. 123:3048-54 |
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| Abstract |
The local electrostatic environment plays a critical role in determining the physicochemical properties of reactive radicals in proteins. High-field electron paramagnetic resonance (HF-EPR) spectroscopy has been used to determine the sensitivity of the tyrosyl radical g-values to local electrostatic environment. Site-specific mutants of ribonucleotide reductase from Escherichia coli were used to study the effect of introducing a charge group on the HF-EPR spectrum of the stable tyrosyl (Y122) radical. The changes affected by the mutations were small, but measurable. Mutation of isoleucine-74 to an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine interactions. 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 shown that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of the nearby diiron center. For the L77F mutant, the shift is induced by the diiron 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. |
| Links | |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Electron Spin Resonance Spectroscopy/methods; Escherichia coli/enzymology; Free Radicals/chemistry; Mutagenesis, Site-Directed; Protein Structure, Quaternary/genetics; Ribonucleotide Reductases/chemistry; Ribonucleotide Reductases/genetics; Tyrosine/chemistry; Tyrosine/genetics |
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