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 t he synthesis of the deoxyribonucleotides required for anaerobic DNA sy nthesis. The enzyme is an alpha(2) beta(2) heterotetramer. in its acti ve form, the large cu,subunit contains are oxygen-sensitive glycyl rad ical, whereas the beta(2) small protein harbors a [4Fe-4S] cluster tha t joins its two polypeptide chains, Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothr eitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (d ithionite or 5-deazaflavin plus light) reducing system. Here, eve demo nstrate that AdoMet is directly reduced by the Fe-S center of beta(2) during the activation of the enzyme, resulting in methionine and glycy l radical formation, Direct binding experiments showed that AdoMet bin ds to beta(2) with a K-d of 10 mu M and a 1:1 stoichiometry Binding wa s confirmed by EPR spectroscopy that demonstrated the formation of a c omplex between AdoMet and the [4Fe-4S] center of beta 2. Dithiothreito l triggered the cleavage of AdoMet, leading to an EPR-silent form of b eta(2) and, in the case of alpha(2) beta(2), to glycyl radical formati on, In both instances, 3 methionines were formed per mol of protein, O ur results indicate that the Fe-S center of beta(2) is directly involv ed in the reductive cleavage of AdoMet and suggest a mew biological fu nction for an iron-sulfur center, i.e redox catalysis, as recently pro posed 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).