Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme Δ9 stearoyl-acyl carrier protein desaturase: Implications for oxygen activation and catalytic intermediates

Δ9 stearoyl-acyl carrier protein (ACP) desaturase is a μ-oxo-bridged di-iron enzyme, which belongs to the structural class I of large helix bundle proteins and that catalyzes the NADPH and O ₂-dependent formation of a cis-double bond in stearoyl-ACP. The crystal structures of complexes with azide and acetate, respectively, as well as the apo- and single-iron forms of Δ9 stearoyl-ACP desaturase from Ricinus communis have been determined. In the azide complex, the ligand forms a μ-1,3-bridge between the two iron ions in the active site, replacing a loosely bound water molecule. The structure of the acetate complex is similar, with acetate bridging the di-iron center in the same orientation with respect to the di-iron center. However, in this complex, the iron ligand Glu ¹⁹⁶ has changed its coordination mode from bidentate to monodentate, the first crystallographic observation of a carboxylate shift in Δ9 stearoyl-ACP desaturase. The two complexes are proposed to mimic a μ-1,2 peroxo intermediate present during catalytic turnover. There are striking structural similarities between the di-iron center in the Δ9 stearoyl-ACP desaturase-azide complex and in the reduced rubrerythrin-azide complex. This suggests that Δ9 stearoyl-ACP desaturase might catalyze the formation of water from exogenous hydrogen peroxide at a low rate. From the similarity in iron center structure, we propose that the μ-oxo-bridge in oxidized desaturase is bound to the di-iron center as in rubrerythrin and not as reported for the R2 subunit of ribonucleotide reductase and the hydroxylase subunit of methane monooxygenase. The crystal structure of the one-iron depleted desaturase species demonstrates that the affinities for the two iron ions comprising the di-iron center are not equivalent, Fe1 being the higher affinity site and Fe2 being the lower affinity site.