Moënne-Loccoz P, Baldwin J, Ley B A, Loehr T M, Bollinger J M
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000.
Biochemistry. 1998 Oct 20;37(42):14659-63. doi: 10.1021/bi981838q.
Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O-O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 --> Glu and the surface mutation Trp48 --> Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe-O2-Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Delta9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664-14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.
非血红素二铁簇存在于多种酶中（如核糖核苷酸还原酶、甲烷单加氧酶和Δ9-硬脂酰-ACP去饱和酶），这些酶能激活氧气以进行化学性质上困难的氧化反应。在每种情况下，人们认为当氧气与二价铁酶反应时会形成动力学不稳定的过氧中间体，随后发生O - O键断裂并形成高价铁中间体[形式上为Fe(IV)]，这些中间体被认为是活性氧化剂。通过铁配体突变Asp84→Glu和表面突变Trp48→Phe，核糖核苷酸还原酶的蛋白质R2中过氧二铁(III)中间体的动力学稳定性得到了提高。在此，我们通过对该突变体R2的冷冻捕获中间体进行振动光谱研究，首次给出了桥连对称过氧加合物的确切证据。在870、499和458 cm-1处观察到对同位素敏感的谱带，分别归属于配体内过氧伸缩频率以及不对称和对称的Fe - O2 - Fe伸缩频率。在对Δ9-硬脂酰-ACP去饱和酶的过氧二铁物种进行的共振拉曼光谱研究中也得到了类似结果[Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664 - 14671]。这些加合物与甲烷单加氧酶羟化酶、铁蛋白和野生型蛋白质R2在氧气激活过程中检测到的瞬态物种之间的相似性表明，对称过氧加合物是这一类成员介导的多种氧化反应中的常见中间体。
