title: Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis.
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 Fe(3+)/Fe(2+) 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, hydroxide-bound 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+)/Fe(2+) reduction potential of H303A variant is approximately 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.
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, United Kingdom.
authors.forename: Nektaria D
authors.initials: ND
authors.lastname: Papadopoulou
authors.forename: Martin
authors.initials: M
authors.lastname: Mewies
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Harriet E
authors.initials: HE
authors.lastname: Seward
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Emma Lloyd
authors.initials: EL
authors.lastname: Raven
mesh_terms: D000409:Alanine / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D014644:Genetic Variation; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013696:Temperature
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008024:Ligands; D006418:Heme; D006639:Histidine; D000409:Alanine