Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex.

Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of v(s)(Fe-O-Fe) and v(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from similar to 123 degrees in the ore-bridged diferric centers of the as-isolated enzyme to similar to 120 degrees in ore-bridged diferric centers of the complex. Analysis of the shifts in v(s)(Fe-O-Fe) and v(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta 9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta 9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta 9D was used to investigate whether an O-atom from O-2 would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of similar to 70% yield of 18:1-ACP product in the presence of O-18(2), no incorporation of an O-18 atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O-2 into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta 9D catalysis are discussed.