Dioxygen is the source of the mu-oxo bridge in iron ribonucleotide reductase.
The formation of the iron-radical cofactor in the R2 subunit of ribonucleotide reductase has been monitored by resonance Raman spectroscopy. The differrous cluster in reduced R2 functions as a tyrosine oxidase; it uses O2 to oxidize Tyr-122 to a stable radical and results in an oxo-bridged diferric cluster. The Phe-122 mutant produces an identical dinuclear iron center and provides a simplified model for O2 activation. Oxidation with 18O2 results in quantitative incorporation of 18O into the diferric cluster as evidenced by the 13-cm-1 downshift in the Fe-O-Fe stretching vibration at 500 cm-1. Thus, O2 must be coordinated to the diiron center during O-O bond cleavage. When the Phe-208 adjacent to the diferous cluster is mutated to Tyr, reaction with O2 results in its oxidation to dihydroxyphenylalanine (DOPA-208) and subsequent coordination to Fe as a catecholate ligand. The Fe-O/(catecholate) stretching modes at 512 and 592 cm-1 shift by -13 and -8 cm-1, respectively, when the oxidation is performed in H(2)18O. These isotope shifts indicate that the second oxygen atom of DOPA-208 originates from H2O rather than O2. Taken together, our results are consistent with a mu-1,1-peroxide and a high valent iron-oxo species as reaction intermediates. A common pathway for oxygen activation by the related iron-oxo enzymes methane monooxygenase and fatty acid desaturase is proposed.
Ling
J
J
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000.
Sahlin
M
M
Sjöberg
B M
BM
Loehr
T M
TM
Sanders-Loehr
J
J
D002396:Catechols / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000737:chemistry*; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics; D013059:Spectrum Analysis, Raman
D002396:Catechols; D007501:Iron; D012264:Ribonucleotide Reductases; D010100:Oxygen