O2 Activation by Non-Heme Diiron Proteins: Identification of a Symmetric μ-1,2-Peroxide in a Mutant of Ribonucleotide Reductase

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Abstract
Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Δ9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O−O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 → Glu and the surface mutation Trp48 → Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe−O2−Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Δ9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry37, 14664−14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.