Combined optical and resonance Raman studies have revealed the formation of an O2-adduct upon exposure of 4e- chemically reduced stearoyl-acyl carrier protein Delta9 desaturase to stearoyl-ACP and 1 atm O2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t1/2 approximately 26 min). Resonance Raman studies using 16O2 gas reveal vibrational features of a bound peroxide [Vs(Fe-O2), 442 cm-1; Vas(Fe-O2), 490 cm-1; V(O-O), 898 cm-1] that undergo the expected mass-dependent shifts when prepared in (16)O(18)O or 18(O2). The appearance of two Fe-O2 vibrations, each having a single peak of intermediate frequency with 16(O)18(O), provs that the peroxide is bound symmetrically between the two iron atoms in a mu-1,2 configuration. The same results have been obtained in the accompanying resonance Raman study of ribonucleotide reductase isoform W48F/D84E [P. Moënne-Loccoz, J. Baldwin, B. A. Ley, T. M. Loehr, and J. M. Bollinger, Jr. (1998) Biochemistry 37, 14659-14663], thus making it likely that other members of the class II diiron enzymes form related peroxodiferric intermediates. Study of the reactivity of peroxodiferric Delta9D revealed that this intermediate underwent 2e- reduction leading to an oxidase reaction and recovery of the resting ferric homodimer. In contrast, biological reduction of the same enzyme preparations using ferredoxin reductase and [2Fe-2S] ferredoxin gave catalytic desaturation with a turnover number of 20-30 min-1. The profound difference in catalytic outcome for chemically and enzymatically reduced Delta9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta9D oxidase reaction represents a new type of reactivity for the acyl-ACP desaturases and provides a two-step catalytic precedent for the "alternative oxidase" activity recently proposed for a membrane diiron enzyme in plants and trypanosomes.