The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center

• Published in: The Journal of biological chemistry 1996-04, Vol.271 (16), p.9410-9416
• Main Authors: Ollagnier, S, Mulliez, E, Gaillard, J, Eliasson, R, Fontecave, M, Reichard, P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anaerobiosis; Centrifugation, Density Gradient; Chromatography, Gel; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli - enzymology; Glycine; Iron - metabolism; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - isolation & purification; Iron-Sulfur Proteins - metabolism; Kinetics; Macromolecular Substances; Medicin och hälsovetenskap; Molecular Weight; Ribonucleotide Reductases - chemistry; Ribonucleotide Reductases - isolation & purification; Ribonucleotide Reductases - metabolism; Sulfides - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.16.9410

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.