Engineering of the iron site in ribonucleotide reductase to a self-hydroxylating monooxygenase.
Protein R2 of ribonucleotide reductase contains a dinuclear ferric iron center adjacent to a tyrosyl radical in the interior of the protein matrix. A patch of hydrophobic residues surrounds the iron-radical cofactor. Its importance during the oxidative generation of the iron-radical cofactor was investigated by site-directed mutagenesis of Phe-208 to tyrosine. The mutant protein R2 F208Y has prominent absorption bands at 460 and 720 nm reminiscent of those in ferric-catecholate complexes. Resonance Raman spectroscopy shows that the iron center of R2 F208Y contains a bidentate catechol ligand. The mechanism for generation of this protein-derived dihydroxyphenylalanine may be similar to the catalytic cycle of methane monooxygenase.
Ormö
M
M
Department of Molecular Biology, Stockholm University, Sweden.
deMaré
F
F
Regnström
K
K
Aberg
A
A
Sahlin
M
M
Ling
J
J
Loehr
T M
TM
Sanders-Loehr
J
J
Sjöberg
B M
BM
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics*; D010649:Phenylalanine / Q000235:genetics; D015202:Protein Engineering; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000235:genetics
D014443:Tyrosine; D010649:Phenylalanine; D007501:Iron; D010105:Oxygenases; D012264:Ribonucleotide Reductases