Peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase:: Oxidase reactivity during single turnover and implications for the mechanism of desaturation

Combined optical and resonance Raman studies have revealed the formation of an Op-adduct upon exposure of 4e(-) chemically reduced stearoyl-acyl carrier protein Delta(9) desaturase to stearoyl-AGP and 1 atm O-2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t(1/2) approximate to 26 min). Resonance Raman studies using O-16(2) gas reveal vibrational features of a bound peroxide [v(s)(Fe-O-2), 442 cm(-1); v(as)(Fe-O-2), 490 cm(-1); v(O-O), 898 cm(-1)] that undergo the expected mass-dependent shifts when prepared in (OO)-O-16-O-18 or O-18(2.) The appearance of two Fe-O-2 vibrations, each having a single peak of intermediate frequency with (OO)-O-16-O-18, proves 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. Moenne-Loccoz, J. Baldurin, 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 Delta 9D 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 Delta 9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta 9D 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.