PEROXODIFERRIC INTERMEDIATE OF STEAROYL-ACYL CARRIER PROTEIN DELTA(9)DESATURASE - OXIDASE REACTIVITY DURING SINGLE TURNOVER AND IMPLICATIONS FOR THE MECHANISM OF DESATURATION

Combined optical and resonance Raman studies have revealed the formati on 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 featur es of a bound peroxide [v(s)(Fe-O-2), 442 cm(-1); v(as)(Fe-O-2), 490 c m(-1); v(O-O), 898 cm(-1)] that undergo the expected mass-dependent sh ifts 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 frequenc y 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 result s have been obtained in the accompanying resonance Raman study of ribo nucleotide 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 clas s II diiron enzymes form related peroxodiferric intermediates. Study o f the reactivity of peroxodiferric Delta 9D revealed that this interme diate underwent 2e(-) reduction leading to an oxidase reaction and rec overy of the resting ferric homodimer. In contrast, biological reducti on 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 chemic ally and enzymatically reduced Delta 9D suggests that redox-state depe ndent conformational changes cause partition of reactivity between des aturase and oxidase chemistries. The Delta 9D oxidase reaction represe nts a new type of reactivity for the acyl-ACP desaturases and provides a two-step catalytic precedent for the ''alternative oxidase'' activi ty recently proposed for a membrane diiron enzyme in plants and trypan osomes.