An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli.
Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase. The enzyme requires anaerobic activation by two E. coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl. We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH. The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster. In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content. The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical. The signal disappeared after exposure of the activated enzyme to air. We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine. From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.
Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Université Joseph Fourier, URA Centre National de la Recherche Scientifique 332, Chimie-Recherche, Grenoble, France.
E
E
Mulliez
M
M
Fontecave
J
J
Gaillard
P
P
Reichard
D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D012436:S-Adenosylmethionine; D012264:Ribonucleotide Reductases