The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center.
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.
Ollagnier
S
S
Laboratoire d'Etudes Dynamiques et Structurales de la Selectivite, Universite Joseph Fourier, Grenoble, France.
Mulliez
E
E
Gaillard
J
J
Eliasson
R
R
Fontecave
M
M
Reichard
P
P
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013440:Sulfides / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D013440:Sulfides; D007501:Iron; D012264:Ribonucleotide Reductases; D005998:Glycine