During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.

Escherichia coli contains a specific ribonucleotide reductase for deoxyribonucleotide synthesis and growth under anaerobiosis. The R 2 β 2 enzyme contains an iron-sulfur center on its small β 2 subunit that is involved in the one-electron reduction of S-adenosylmethionine and in the generation of a glycyl radical on the large R 2 subunit. By a variety of spectroscopic methods (light absorption, resonance Raman, and Mo ¨ssbauer spectroscopy) and metal and sulfide analysis, it is shown that the metal center is a (2Fe-2S) 2+ cluster. Reduction by photoreduced deazaflavin or dithionite converts these centers mostly into (4Fe-4S) clusters, in both the (4Fe-4S) 1+ and (4Fe-4S) 2+ states, as is unambiguously demonstrated by Mo ¨ssbauer spectroscopy at 4.2 and 77 K, in the presence of small (10 mT) or high (5.3 or 7 T) fields. The structure and the function of the iron-sulfur center of the anaerobic ribonucleotide reductase are discussed in relation with other members of a class of enzymes with similar metal centers and functions (reduction of S-adenosylmethionine).