Binding of Imidazole Stabilizes Low‐spin State of Heme Iron in Dual‐Substrate‐Specific Rice Allene Oxide Synthase‐1

Abstract

Kinetic studies have indicated that imidazole is a competitive inhibitor of rice allene oxide synthase-1 with K i of 34.9, 14.3, 40.3, and 23.3 mM for 13(S)-HPODE, 13(S)-HPOTE, 9(S)-HPODE, and 9(S)-HPOTE as substrates, respectively. Imidazole-free rice allene oxide synthase-1 has a Soret absorbance maximum at 393 nm and occupied high-spin (g = 6.0) and low-spin ( g = 2.39, 2.24, 1.93) states in the ratio 1:10, as detected by EPR spectroscopy, while in the presence of imidazole the occupancy of the low-spin state increased to 100%. The results support the hypothesis that imidazole competes with the substrate of allene oxide synthase-1 for binding to the sixth position of heme iron(III) and that binding of imidazole stabilizes the low-spin state. The role of detergent binding and implications for the mechanism by which allene oxide synthase-1 cleaves the RO-OH bond are discussed.