Department of Life Science and Picobiology Institute, Graduate School of Life Science, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
Biochemistry. 2010 Nov 30;49(47):10081-8. doi: 10.1021/bi1009997. Epub 2010 Nov 8.
Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme which catalyzes dioxygenation of l-Trp (tryptophan), yielding N-formylkynurenine. IDO thus plays a key role in l-Trp catabolism in mammals. In the present study, resonance Raman (RR) spectra of the reduced carbon monoxide- (CO-) bound form of IDO were measured in order to gain insights into the active site environment of O(2). Binding of CO to l-Trp-bound IDO causes a significant change in the electronic and RR spectra of the heme, indicating that the π* orbitals of the carbon atom of CO interact with π orbitals of Fe and the porphyrin. On the other hand, binding of CO to d-Trp-bound IDO does not induce the same change. This is also the case with substrate-free IDO. Based on the distinct absorption spectra and RR bands of the vibrational signature of CO (ν(CO), δ(FeCO), and ν(Fe-CO)) of the l-Trp-bound species relative to the other two species, it is confirmed that sterically constrained geometry of the Fe-O-O unit exists as previously reported (Terentis, A. C., et al. (2002) J. Biol. Chem. 277, 15788-15794). In contrast, binding of d-Trp does not induce such constraint. The comparable values of V(max) reported for l-Trp and d-Trp are interpreted as a result of a change in the rate-limiting step in the reaction cycle of the enzyme induced by the d-enantiomer relative to the l-enantiomer. Enhancements of the overtone and the combination Raman modes of the Fe-CO stretching vibration are evident. The anharmonicity of the Fe-CO stretching oscillator is significantly higher than those of oxygen carrier proteins. This is a specific character of IDO and might be responsible for the unique reactivity of this enzyme.
吲哚胺 2,3-双加氧酶 (IDO) 是一种血红素酶，可催化 l-Trp（色氨酸）的双加氧作用，生成 N-甲酰犬尿氨酸。因此，IDO 在哺乳动物中 l-Trp 分解代谢中起着关键作用。在本研究中，测量了还原的一氧化碳-（CO-）结合形式 IDO 的共振拉曼（RR）光谱，以便深入了解 O(2)的活性位点环境。CO 与 l-Trp 结合的 IDO 结合会导致血红素的电子和 RR 光谱发生显著变化，表明 CO 碳原子的 π*轨道与 Fe 和卟啉的 π 轨道相互作用。另一方面，CO 与 d-Trp 结合的 IDO 不会引起相同的变化。对于无底物的 IDO 也是如此。基于 l-Trp 结合物相对于其他两种物质的 CO（ν(CO)、δ(FeCO)和 ν(Fe-CO))的振动特征的独特吸收光谱和 RR 带，确认先前报道的 Fe-O-O 单元的空间受限几何形状存在（Terentis，A. C.，等。（2002）J. Biol. Chem. 277, 15788-15794）。相比之下，d-Trp 的结合不会引起这种限制。报道的 l-Trp 和 d-Trp 的 V(max)值相当，这被解释为由于 d-对映体相对于 l-对映体引起的酶反应循环中的限速步骤发生变化。Fe-CO 伸缩振动的泛音和组合拉曼模式的增强是显而易见的。Fe-CO 伸缩振动的非谐性明显高于氧载体蛋白。这是 IDO 的一个特定特征，可能是该酶独特反应性的原因。
