THE ANAEROBIC ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE - SUBUNIT STRUCTURE AND IRON-SULFUR CENTER

During anaerobic growth Escherichia coil uses a specific ribonucleosid e triphosphate reductase for the production of deoxyribonucleoside tri phosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha(2)) with a stable, oxygen-sensi tive radical located at Gly-681 of the 80-kDa polypeptide chain. The r adical is formed in an enzymatic reaction involving S-adenosylmethioni ne, NADPH, a reducing flavodoxin system and an additional 17.5-kDa pol ypeptide, previously called activase. Here, we demonstrate by EPR spec troscopy 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 ear lier in preparations of the large 160-kDa subunit that suggested the p resence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein, The large and t he small proteins form a tight complex. From sucrose gradient centrifu gation, we determined a 1:1 stoichiometry of the two proteins in the c omplex. The anaerobic reductase thus has an alpha(2) beta(2) structure , We speculate that the small protein interacts with S-adenosylmethion ine and forms a transient radical involved in the generation of the st able glycyl radical in the large protein that participates in the cata lytic process.