Activation of the anaerobic ribonucleotide reductase from Escherichia coli - The essential role of the iron-sulfur center for S-adenosylmethionine reduction

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha(2) beta(2) heterotetramer. in its active form, the large cu,subunit contains are oxygen-sensitive glycyl radical, whereas the beta(2) small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains, Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, eve demonstrate that AdoMet is directly reduced by the Fe-S center of beta(2) during the activation of the enzyme, resulting in methionine and glycyl radical formation, Direct binding experiments showed that AdoMet binds to beta(2) with a K-d of 10 mu M 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 beta 2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta(2) and, in the case of alpha(2) beta(2), to glycyl radical formation, In both instances, 3 methionines were formed per mol of protein, Our results indicate that the Fe-S center of beta(2) is directly involved in the reductive cleavage of AdoMet and suggest a mew 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,, Schurmann, P,, Knaff D, B,, and Johnson, M, K, (1996) Biochemistry 35, 11425-11434).