Peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase:: Oxidase reactivity during single turnover and implications for the mechanism of desaturation
Peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase:: Oxidase reactivity during single turnover and implications for the mechanism of desaturation
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DOI：
10.1021/bi981839i
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发表时间：
1998-10-20
期刊：
影响因子：
2.9
通讯作者：
Fox, BG
中科院分区：
文献类型：
--
作者：
Broadwater, JA;Ai, JY;Fox, BG
结合光学和共振拉曼研究揭示了在将 4e(-) 化学还原的硬脂酰-酰基载体蛋白 Delta(9) 去饱和酶暴露于硬脂酰-AGP 和 1 atm O-2 时形成 Op 加合物。观察到的中间体在 700 nm 处具有宽吸收带，并且在室温下非常稳定（t(1/2) 约为 26 分钟）。使用 O-16(2) 气体的共振拉曼研究揭示了结合过氧化物的振动特征 [v(s)(Fe-O-2), 442 cm(-1)； v(as)(Fe-O-2), 490 厘米(-1); v(O-O), 898 cm(-1)] 在 (OO)-O-16-O-18 或 O-18(2.) 中制备时经历预期的质量依赖位移。两个 Fe-O-2 振动的出现，每个振动与 (OO)-O-16-O-18 具有中频单峰，证明过氧化物在 mu-1,2 构型中对称地结合在两个铁原子之间。在核糖核苷酸还原酶异构体 W48F/D84E 的伴随共振拉曼研究中也获得了相同的结果 [P. Moenne-Loccoz, J. Baldurin, B. A. Ley, T. M. Loehr, and J. M. Bollinger, Jr. (1998) Biochemistry 37, 14659-14663]，因此使得 II 类二铁酶的其他成员可能形成相关的过氧二铁中间体。对过氧化二铁 Delta 9D 反应性的研究表明，该中间体经历了 2e(-) 还原，导致氧化酶反应并恢复静止的三价铁同二聚体。相比之下，使用铁氧还蛋白还原酶和[2Fe-2S]铁氧还蛋白对相同酶制剂进行生物还原，产生催化去饱和，周转数为20-30分钟(-1)。化学和酶还原 Delta 9D 催化结果的巨大差异表明氧化还原态依赖性构象变化导致去饱和酶和氧化酶化学之间的反应性划分。 Delta 9D 氧化酶反应代表了酰基-ACP 去饱和酶的一种新型反应性，并为最近提出的植物和锥虫膜二铁酶的“替代氧化酶”活性提供了两步催化先例。
Combined optical and resonance Raman studies have revealed the formation of an Op-adduct upon exposure of 4e(-) chemically reduced stearoyl-acyl carrier protein Delta(9) desaturase to stearoyl-AGP and 1 atm O-2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t(1/2) approximate to 26 min). Resonance Raman studies using O-16(2) gas reveal vibrational features of a bound peroxide [v(s)(Fe-O-2), 442 cm(-1); v(as)(Fe-O-2), 490 cm(-1); v(O-O), 898 cm(-1)] that undergo the expected mass-dependent shifts when prepared in (OO)-O-16-O-18 or O-18(2.) The appearance of two Fe-O-2 vibrations, each having a single peak of intermediate frequency with (OO)-O-16-O-18, proves that the peroxide is bound symmetrically between the two iron atoms in a mu-1,2 configuration. The same results have been obtained in the accompanying resonance Raman study of ribonucleotide reductase isoform W48F/D84E [P. Moenne-Loccoz, J. Baldurin, B. A. Ley, T. M. Loehr, and J. M. Bollinger, Jr. (1998) Biochemistry 37, 14659-14663], thus making it likely that other members of the class II diiron enzymes form related peroxodiferric intermediates. Study of the reactivity of peroxodiferric Delta 9D revealed that this intermediate underwent 2e(-) reduction leading to an oxidase reaction and recovery of the resting ferric homodimer. In contrast, biological reduction of the same enzyme preparations using ferredoxin reductase and [2Fe-2S] ferredoxin gave catalytic desaturation with a turnover number of 20-30 min(-1). The profound difference in catalytic outcome for chemically and enzymatically reduced Delta 9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta 9D oxidase reaction represents a new type of reactivity for the acyl-ACP desaturases and provides a two-step catalytic precedent for the "alternative oxidase" activity recently proposed for a membrane diiron enzyme in plants and trypanosomes.