EXAFS and Mössbauer characterization of the Diiron(III) site in stearoyl-acyl carrier protein Δ9– desaturase

Abstract

Stearoyl-acyl carrier protein (ACP) Δ9-desaturase (Δ9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O2- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation state. These studies reveal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41 A. The species having the 3.12 A Fe-Fe distance also exhibits a 1.8 A Fe-O bond and is thus proposed to represent Δ9D molecules containing a (μ-oxo)bis(μ-carboxylato)diiron(III) cluster. The species having the 3.41 A Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Δ9D molecules containing a (μ-hydroxo)diiron(III) cluster. Mossbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (ΔEQ(1)=1.53 mm/s, 72%;ΔEQ(2)=0.72 mm/s, 21%;ΔEQ(3)=2.20 mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (μ-oxo)- and (μ-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mossbauer majority species, is likely associated with the EXAFS majority species having a 3.12 A Fe-Fe separation and a 1.8 A Fe-μ-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41 A Fe-Fe separation. The presence of multiple diiron center conformations in diferric Δ9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (∼9 kDa) during desaturation catalysis.