EPR and Mossbauer Spectroscopy Show Inequivalent Hemes in Tryptophan Dioxygenase
EPR and Mossbauer Spectroscopy Show Inequivalent Hemes in Tryptophan Dioxygenase
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DOI：
10.1021/ja908851e
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发表时间：
2010-01-27
影响因子：
15
通讯作者：
Hendrich, Michael P.
中科院分区：
文献类型：
--
作者：
Gupta, Rupal;Fu, Rong;Hendrich, Michael P.
色氨酸2,3-二加氧酶（TDO）是NAD生物合成途径中的必不可少的酶，对所有生物体都很重要。 TDO催化L-色氨酸（L-TRP）的吲哚环的氧化裂解，将其转化为N-纤维基氨瑞宁（NFK）。 TDO的晶体结构显示出同倍跨蛋白的二聚体四二聚体结构的二聚体。该蛋白质的四个催化位点，每个亚基一个含有血红素，可催化二恶英的激活和插入到L-TRP中。由于具有α（4）结构，并且在以前的光谱研究中仅确定了一种类型的血红素中心，因此假定这四个HEMES位点是等效的。目前的工作表明，TDO的血红素位点不是等效的。 EPR和Mossbauer光谱数据的定量解释表明，在酶的还原和氧化状态中存在两个主要的不相等性血红素物种，这与现在延伸至血液电子特性的二聚体蛋白质季季结构的二聚体一致。 L-TRP添加后，HEMES在降低的TDO状态下的电子特性显着变化，这归因于近端组氨酸的质子化状态变化，向Hemes。 02替代的结合NO或CO显示了两个不等的血红素位点。血红素-NO复合物为5和6坐标，没有L-TRP，并且都与L-TRP进行6坐标。只能从一个血红素-NO位点中选择性地分离，只能在L-TRP的存在下进行散热。 TDO的冷冻度产生了一种新型的Diamagnetic Heme物种，暂定地分配为减少的Heme-OH复合物。这项工作介绍了与蛋白质的血红素相互作用的新描述，并在其近端他的近端中进行了描述，在对物理数据的一般解释中必须考虑与TDO的动力学，机制和功能有关。
Tryptophan 2,3-dioxygenase (TDO) is an essential enzyme in the pathway of NAD biosynthesis and important for all living organisms. TDO catalyzes oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The crystal structure of TDO shows a dimer of dimer quaternary structure of the homotetrameric protein. The four catalytic sites of the protein, one per subunit, contain a heme that catalyzes the activation and insertion of dioxygen into L-Trp. Because of the alpha(4) structure and because only one type of heme center has been identified in previous spectroscopic studies, the four hemes sites have been presumed to be equivalent. The present work demonstrates that the heme sites of TDO are not equivalent. Quantitative interpretation of EPR and Mossbauer spectroscopic data indicates the presence of two dominant inequivalent heme species in reduced and oxidized states of the enzyme, which is consistent with a dimer of dimer protein quaternary structure that now extends to the electronic properties of the hemes. The electronic properties of the hemes in the reduced state of TDO change significantly upon L-Trp addition, which is attributed to a change in the protonation state of the proximal histidine to the hemes. The binding Of 02 surrogates NO or CO shows two inequivalent heme sites. The heme-NO complexes are 5- and 6-coordinate without L-Trp, and both 6-coordinate with L-Trp. NO can be selectively photodissociated from only one of the heme-NO sites and only in the presence Of L-Trp. Cryoreduction of TDO produces a novel diamagnetic heme species, tentatively assigned as a reduced heme-OH complex. This work presents a new description of the heme interactions with the protein, and with the proximal His, which must be considered during the general interpretation of physical data as it relates to kinetics, mechanism, and function of TDO.