THE CONSERVED SERINE-211 IS ESSENTIAL FOR REDUCTION OF THE DINUCLEAR IRON CENTER IN PROTEIN R2 OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE

The R2 protein family of class I ribonucleotide reductases contains a highly conserved serine residue close to the essential tyrosyl radical and the dinuclear iron center. In order to test its physiological imp ortance, we have engineered the Ser-211 of Escherichia coli R2 to an a lanine and a cysteine residue. The three-dimensional structure of R2 S 211A solved to 2.4-Angstrom resolution is virtually identical to the w ild-type structure apart from the substituted residue. Both mutant pro teins contain oxidized dinuclear iron and tyrosyl radical, and their s pecific enzyme activity per radical are comparable to that of the wild -type protein. In R2 S211A the stability of the tyrosyl radical is sub stantially decreased, probably caused by movement of Gln-80 into hydro gen bonding distance of Tyr-122. The major defect in R2 S211A, however , is the inability of its iron center to be reduced by enzymic or chem ical means, a characteristic not found in R2 S211C. We propose that Se r-211 is needed as a proton donor/transporter during reduction of the iron center of R2, a reaction which in vivo precedes reconstitution of the tyrosyl radical. This offers a physiological explanation for the high conservation of a serine residue at this position in the R2 famil y.