| Abstrakt: |
The anaerobic ribonucleotide reductase ofEscherichia colicatalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an α2β2heterotetramer. In its active form, the large α2subunit contains an oxygen-sensitive glycyl radical, whereas the β2small 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 β2during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to β2with aKdof 10 μmand 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 β2and, 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 β2is 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) Biochemistry35, 11425–11434). |
