Broadwater J A, Ai J, Loehr T M, Sanders-Loehr J, Fox B G
The Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705, USA.
Biochemistry. 1998 Oct 20;37(42):14664-71. doi: 10.1021/bi981839i.
Combined optical and resonance Raman studies have revealed the formation of an O2-adduct upon exposure of 4e- chemically reduced stearoyl-acyl carrier protein Delta9 desaturase to stearoyl-ACP and 1 atm O2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t1/2 approximately 26 min). Resonance Raman studies using 16O2 gas reveal vibrational features of a bound peroxide [Vs(Fe-O2), 442 cm-1; Vas(Fe-O2), 490 cm-1; V(O-O), 898 cm-1] that undergo the expected mass-dependent shifts when prepared in (16)O(18)O or 18(O2). The appearance of two Fe-O2 vibrations, each having a single peak of intermediate frequency with 16(O)18(O), provs 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. Moënne-Loccoz, J. Baldwin, 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 Delta9D 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 Delta9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta9D 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.
结合光学和共振拉曼光谱研究表明，当化学还原的硬脂酰 - 酰基载体蛋白Δ9去饱和酶（4e-）暴露于硬脂酰 - ACP和1个大气压的O2时，会形成O2加合物。观察到的中间体在700 nm处有一个宽吸收带，并且在室温下非常稳定（半衰期约为26分钟）。使用16O2气体进行的共振拉曼研究揭示了结合过氧化物的振动特征[Vs(Fe - O2)，442 cm-1；Vas(Fe - O2)，490 cm-1；V(O - O)，898 cm-1]，当在(16)O(18)O或18(O2)中制备时，这些特征会发生预期的质量依赖性位移。出现两个Fe - O2振动，每个在16(O)18(O)时都有一个中频单峰，这证明过氧化物以μ - 1,2构型对称地结合在两个铁原子之间。在核糖核苷酸还原酶同工型W48F/D84E的伴随共振拉曼研究中也得到了相同的结果[P. Moënne - Loccoz，J. Baldwin，B. A. Ley，T. M. Loehr，和J. M. Bollinger，Jr.（1998年）《生物化学》37卷，14659 - 14663页]，因此很可能II类双铁酶的其他成员也会形成相关的过氧二铁中间体。对过氧二铁Δ9D反应性的研究表明，该中间体经历2e-还原，导致氧化酶反应并恢复静止的铁同二聚体。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时，催化去饱和的周转数为20 - 30 min-1。化学还原和酶促还原的Δ9D在催化结果上的巨大差异表明，氧化还原状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分配。Δ9D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中膜双铁酶的“替代氧化酶”活性提供了一个两步催化先例。
