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

The stearoyl-acyl carrier protein Delta(9) desaturase (Delta(9)D) uses an ore-bridged diiron center to cata lyze the NAD(P)H- and O-2-dependent desaturation of stearoyl-ACP. Delta 9D, 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 O-2 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 Delta 9D 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 (k(app) approximate to 3.5 s(-1) in 0.7 M NaN3, pH 7.8). RR experiments demonstrate the existence of two different adducts: an eta(1)-terminal structure at pH 7.8 (N-14(3)- asymmetric stretch at 2073 cm(-1), resolved into two bands with (NN2-)-N-15-N-14) and a mu-1,3 bridging structure at pH < 7 (N-14(3)- asymmetric stretch at 2100 cm(-1), shifted as a single band with (NN2-)-N-15-N-14). Both adducts also exhibit an Fe-N-3 stretching mode at approximate to 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 mu-1,3 bridging azide supports the likelihood of a mu-1,2 bridging peroxide as a catalytic intermediate in the Delta 9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries.