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 ribonu cleotide reductase has been monitored by resonance Raman spectroscopy. The diferrous cluster in reduced R2 functions as a tyrosine oxidase; it uses O-2 to oxidize Tyr-122 to a stable radical and results in an o re-bridged diferric cluster. The Phe-122 mutant produces an identical dinuclear iron center and provides a simplified model for O-2 activati on. Oxidation with O-18(2) results in quantitative incorporation of O- 18 into the diferric cluster as evidenced by the 13-cm(-1) downshift i n the Fe-O-Fe stretching vibration at 500 cm(-1). Thus, O-2 must be co ordinated to the diiron center during O-O bond cleavage. When the Phe- 208 adjacent to the diferrous cluster is mutated to Tyr, reaction with O-2 results in its oxidation to dihydroxyphenylalanine (DOPA-208) and subsequent coordination to Fe as a catecholate ligand. The Fe-O/(cate cholate) stretching modes at 512 and 592 cm(-1) shift by -13 and -8 cm (-1), respectively, when the oxidation is performed in (H2O)-O-18, The se isotope shifts indicate that the second oxygen atom of DOPA-208 ori ginates from H2O rather than O-2. Taken together, our re suits are con sistent with a mu-1,1-peroxide and a high valent iron-ore species as r eaction intermediates. A common pathway for oxygen activation by the r elated iron-ore enzymes methane monooxygenase and fatty acid desaturas e is proposed.