Redox and Spectroscopic Properties of Human Indoleamine 2,3-Dioxygenase and A His303Ala Variant: Implications for Catalysis

Nektaria D. Papadopoulou, Martin Mewies, Kirsty J. McLean, Harriet E. Seward, Dimitri A. Svistunenko, Andrew W. Munro, Emma Lloyd Raven

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65 Citations (Scopus)

Abstract

Indoleamine 2,3-dioxygenase is an important mammalian target that catalyses the oxidative cleavage of L-tryptophan to N-formylkynurenine. In this work, the redox properties of recombinant human indoleamine 2,3-dioxygenase (rhIDO) and its H303A variant have been examined for the first time and the spectroscopic and substrate-binding properties of rhIDO and H303A in the presence and absence of substrate are reported. The Fe3+TFe2+ reduction potential of H303A was found to be -30 ± 4 mV; in the presence of L-Trp, this value increases to +16 ± 3 mV. A variety of spectroscopies indicate that ferric rhIDO at pH 6.6 exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin species that contains a second nitrogenous ligand; parallel experiments on H303A are consistent either with His303 as the sixth ligand or with His303 linked to a conformational change that affects this transition. There is an increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-bound heme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxidebound heme; analysis of the H303A variant indicates that His303 is not required for this conversion and is not essential for substrate binding. The Fe 3+TFe2+ reduction potential of H303A variant is ≈70 mV lower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligate intermediate in the catalytic process. In comparison with the properties of other heme enzymes, the data can be used to build a more detailed picture of substrate binding and catalysis in indoleamine 2,3-dioxygenase. The wider implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme.

Original languageEnglish
Pages (from-to)14318-14328
Number of pages11
JournalBiochemistry
Volume44
Issue number43
Early online date5 Oct 2005
DOIs
Publication statusPublished - 1 Nov 2005
Externally publishedYes

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