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 Δ9 desaturase. These studies show that complex formation produces changes in the frequencies of ν(s)(Fe-O-Fe) and ν(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from ~ 123°in the oxo-bridged diferric centers of the as-isolated enzyme to ~ 120° in oxo-bridged diferric centers of the complex. Analysis of the shifts in ν(s)(Fe-O-Fe) and ν(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e--reduced Δ9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Δ9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Δ9D was used to investigate whether an O-atom from O2 would be incorporated into a bridging position of the resultant μ-oxo-bridged diferric centers during the desaturation reaction. Upon formation of ~ 70% yield of 18:1-ACP product in the presence of 18O2, no incorporation of an 18O atom into the μ-oxo bridge position was detected. The result with 18:0-ACP Δ9 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 O2 into the μ-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 Δ9D catalysis are discussed.
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