Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking

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: α2 and β2. As a first step towards mapping the subunit interface, β2 (V365C) was labeled with [14C]-benzophenone (BP) iodoacetamide. The resulting [14C]-BP–β2 (V365C) was complexed with α2 and irradiated at 365 nm for 30 min at 4 °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 [14C]-labeled peptides were identified: two contained a peptide in β to which the BP was attached. The third contained the same β peptide and a peptide in α found in its αD helix. These results provide direct support for the proposed docking model of α2β2.