Abstract
Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [(14)C]-labeled peptides were identified: two contained a peptide in beta to which the BP was attached. The third contained the same beta peptide and a peptide in alpha found in its alphaD helix. These results provide direct support for the proposed docking model of alpha2beta2.