Redox and Spectroscopic Properties of Human Indoleamine 2,3-Dioxygenase and A His303Ala Variant: Implications for Catalysis
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- 作者：Nektaria D. Papadopoulou ; Martin Mewies ; Kirsty J. McLean ; Harriet E. Seward ; Dimitri A. Svistunenko ; Andrew W. Munro ; and Emma Lloyd Raven
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：November 1, 2005
- 年：2005
- 卷：44
- 期：43
- 页码：14318 - 14328
- 全文大小：170K
- 年卷期：v.44,no.43(November 1, 2005)
- ISSN：1520-4995
文摘
Indoleamine 2,3-dioxygenase is an important mammalian target that catalyses the oxidativecleavage of L-tryptophan to N-formylkynurenine. In this work, the redox properties of recombinant humanindoleamine 2,3-dioxygenase (rhIDO) and its H303A variant have been examined for the first time andthe spectroscopic and substrate-binding properties of rhIDO and H303A in the presence and absence ofsubstrate are reported. The Fe3+/Fe2+ reduction potential of H303A was found to be -30 ± 4 mV; in thepresence of L-Trp, this value increases to +16 ± 3 mV. A variety of spectroscopies indicate that ferricrhIDO at pH 6.6 exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin speciesthat contains a second nitrogenous ligand; parallel experiments on H303A are consistent either with His303as the sixth ligand or with His303 linked to a conformational change that affects this transition. There isan increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-boundheme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxide-bound heme; analysis of the H303A variant indicates that His303 is not required for this conversion andis not essential for substrate binding. The Fe3+/Fe2+ reduction potential of H303A variant is 
70 mVlower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligateintermediate in the catalytic process. In comparison with the properties of other heme enzymes, the datacan be used to build a more detailed picture of substrate binding and catalysis in indoleamine2,3-dioxygenase. The wider implications of these results are discussed in the context of our currentunderstanding of the catalytic mechanism of the enzyme.
70 mVlower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligateintermediate in the catalytic process. In comparison with the properties of other heme enzymes, the datacan be used to build a more detailed picture of substrate binding and catalysis in indoleamine2,3-dioxygenase. The wider implications of these results are discussed in the context of our currentunderstanding of the catalytic mechanism of the enzyme.