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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Trapping and spectroscopic characterization of an Fe~(III)-superoxo intermediate from a nonheme mononuclear iron-containing enzyme
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Trapping and spectroscopic characterization of an Fe~(III)-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

机译：非血红素单核含铁酶的Fe〜（III）-超氧中间体的捕集和光谱表征

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摘要

Fe~(III)-O_2~(.-) intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe" enzyme family. Many steps in the O_2 activation and reaction cycle of Fe"-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (lnt-1) observed after adding O_2 to the H200N-4NC complex is trapped and characterized using EPR and Moss-bauer (MB) spectroscopies. lnt-1 is a high-spin (S_1 = 5/2) Fe~(III) anti-ferromagnetically (AF) coupled to an S_2 = 1/2 radical (J≈6 cm~(-1) in H = JS_1 ·S_2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by ~(17)O_2 hyperfine interactions (A_(17O)≈180 MHz). Thus, lnt-1 is an AF-coupled Fe~(III)-O_2~(·-) species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O_2. lnt-1 decays to form a second intermediate (lnt-2). MB spectra show that it is also an AF-coupled Fe~(III)-radkal complex, lnt-2 exhibits an EPR signal at g = 8.05 arising from an 5 = 2 state. The signal is only slightly broadened by ~(17)O_2 (<3% spin delocalization), suggesting that lnt-2 is a peroxo-Fe~(III)-4NC semiquinone radical species. Our results demonstrate facile electron transfer between Fe~(II), O_2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.
机译：Fe〜（III）-O_2〜（.-）中间体在血红素酶中是众所周知的，但在非血红素单核Fe“酶家族中却没有任何特征。在含有Fe”的纯净儿茶酸2的O_2活化和反应循环中的许多步骤通过使用备选底物4-硝基邻苯二酚（4NC）和将活性位点His200突变为Asn（H200N），可以检测到3-3-加氧酶。在这里，使用EPR和Moss-bauer（MB）光谱法捕获并向H200N-4NC络合物中添加O_2后观察到的第一中间体（lnt-1）。 lnt-1是高自旋（S_1 = 5/2）Fe〜（III）反铁磁（AF）耦合到S = 2自由基（H≈JS_1中的J≈6cm〜（-1）） S_2）。它在S = 2多重峰的g = 8.17处以及在S = 3多重峰的g = 8.8和11.6处表现出并行模式EPR信号。这些信号通过〜（17）O_2超精细相互作用（A_（17O）≈180MHz）显着加宽。因此，lnt-1是一种AF耦合的Fe〜（III）-O_2〜（·-）物种。实验观察得到密度泛函理论计算的支持，这些计算表明自旋密度几乎完全转移到结合的O_2上。 lnt-1衰减形成第二个中间体（lnt-2）。 MB光谱表明它也是一种AF耦合的Fe〜（III）-radkal配合物，lnt-2在5 = 2的状态下在g = 8.05时表现出EPR信号。该信号仅被〜（17）O_2稍微加宽（<3％自旋离域），表明lnt-2是一种过氧-Fe〜（III）-4NC半醌自由基物种。我们的结果表明，Fe〜（II），O_2和有机配体之间容易进行电子转移，从而支持提出的野生型酶机制。

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