Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

Abstract

The stearoyl-acyl carrier protein Δ9 desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O2–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O2 to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (kapp≈3.5 s–1 in 0.7 M NaN3, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η1–terminal structure at pH 7.8 (14N3– asymmetric stretch at 2073 cm–1, resolved into two bands with 15N14N2–) and a μ-1,3 bridging structure at pH<7 (14N3– asymmetric stretch at 2100 cm–1, shifted as a single band with 15N14N2–). Both adducts also exhibit an Fe–N3 stretching mode at ≈380 cm–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries.