Activation of the Anaerobic Ribonucleotide Reductase fromEscherichia coli

Abstract

The anaerobic ribonucleotide reductase ofEscherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an α2β2 heterotetramer. In its active form, the large α2 subunit contains an oxygen-sensitive glycyl radical, whereas the β2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requiresS-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of β2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to β2 with aK d of 10 μm and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of β2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of β2 and, in the case of α2β2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of β2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425–11434).