Moche Martin, Shanklin John, Ghoshal Alokesh, Lindqvist Ylva
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm S-171 77, Sweden.
J Biol Chem. 2003 Jul 4;278(27):25072-80. doi: 10.1074/jbc.M301662200. Epub 2003 Apr 18.
Delta9 stearoyl-acyl carrier protein (ACP) desaturase is a mu-oxo-bridged di-iron enzyme, which belongs to the structural class I of large helix bundle proteins and that catalyzes the NADPH and O2-dependent formation of a cis-double bond in stearoyl-ACP. The crystal structures of complexes with azide and acetate, respectively, as well as the apoand single-iron forms of Delta9 stearoyl-ACP desaturase from Ricinus communis have been determined. In the azide complex, the ligand forms a mu-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 Glu196 has changed its coordination mode from bidentate to monodentate, the first crystallographic observation of a carboxylate shift in Delta9 stearoyl-ACP desaturase. The two complexes are proposed to mimic a mu-1,2 peroxo intermediate present during catalytic turnover. There are striking structural similarities between the di-iron center in the Delta9 stearoyl-ACP desaturase-azide complex and in the reduced rubrerythrin-azide complex. This suggests that Delta9 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 mu-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.
Δ9硬脂酰-酰基载体蛋白（ACP）去饱和酶是一种μ-氧桥联双铁酶，属于大螺旋束蛋白的结构I类，催化硬脂酰-ACP中依赖于NADPH和O2的顺式双键形成。已分别测定了蓖麻Δ9硬脂酰-ACP去饱和酶与叠氮化物和乙酸盐的复合物晶体结构，以及该酶的脱辅基和单铁形式的晶体结构。在叠氮化物复合物中，配体在活性位点的两个铁离子之间形成μ-1,3桥，取代了一个松散结合的水分子。乙酸盐复合物的结构与之相似，乙酸盐以相同方向桥联双铁中心。然而，在该复合物中，铁配体Glu196的配位模式从双齿变为单齿，这是在Δ9硬脂酰-ACP去饱和酶中首次通过晶体学观察到的羧酸盐移位。这两种复合物被认为模拟了催化周转过程中存在的μ-1,2过氧中间体。Δ9硬脂酰-ACP去饱和酶-叠氮化物复合物中的双铁中心与还原型红细菌红素-叠氮化物复合物中的双铁中心存在显著的结构相似性。这表明Δ9硬脂酰-ACP去饱和酶可能以低速率催化外源过氧化氢形成水。基于铁中心结构的相似性，我们提出氧化态去饱和酶中的μ-氧桥与双铁中心的结合方式与红细菌红素相同，而不像核糖核苷酸还原酶的R2亚基和甲烷单加氧酶的羟化酶亚基那样。单铁缺失的去饱和酶物种的晶体结构表明，构成双铁中心的两个铁离子的亲和力并不相同，Fe1是高亲和力位点，Fe2是低亲和力位点。
