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 importance, we have engineered the Ser-211 of Escherichia coli R2 to an alanine and a cysteine residue. The three-dimensional structure of R2 S211A solved to 2.4-A resolution is virtually identical to the wild-type structure apart from the substituted residue. Both mutant proteins contain oxidized dinuclear iron and tyrosyl radical, and their specific enzyme activity per radical are comparable to that of the wild-type protein. In R2 S211A the stability of the tyrosyl radical is substantially decreased, probably caused by movement of Gln-80 into hydrogen 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 chemical means, a characteristic not found in R2 S211C. We propose that Ser-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 family.
Regnström
K
K
Department of Molecular Biology, Stockholm University, Sweden.
Aberg
A
A
Ormö
M
M
Sahlin
M
M
Sjöberg
B M
BM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D014961:X-Ray Diffraction
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D007501:Iron; D012264:Ribonucleotide Reductases