Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol.
The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gräslund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.
Fontecave
M
M
Laboratoire d'Etudes Dynamiques de la Structure et de la Sélectivité, Université Joseph Fourier, Grenoble, France.
Gerez
C
C
Mansuy
D
D
Reichard
P
P
D000693:Anaerobiosis; D002374:Catalase / Q000494:pharmacology; D002413:Cations, Divalent; D003676:Deferoxamine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005290:Ferric Compounds / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D002413:Cations, Divalent; D004793:Enzyme Reactivators; D005290:Ferric Compounds; D005296:Ferrous Compounds; D014443:Tyrosine; D004492:Edetic Acid; D002374:Catalase; D013482:Superoxide Dismutase; D012264:Ribonucleotide Reductases; D008274:Magnesium; D003676:Deferoxamine; D004229:Dithiothreitol