Human heme dioxygenases

The L-kynurenine pathway, which leads to the formation of NAD, is the major catabolic route of L-tryptophan metabolism in biology. The initial step in this pathway is oxidation of L-tryptophan to N-formyl-kynurenine. In all biological systems examined to date, this is catalysed by one of two heme enzymes, indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). In this thesis the reaction mechanism, the reactive catalytic intermediates involved in this reaction and the nature of substrate (L-tryptophan and dioxygen)-protein interactions, if any, present within the active site of rhIDO have been examined.;In Chapter 2, we addressed the role of S I67 in rhIDO (S167A and S167H), which is replaced with a histidine residue in TDO enzymes. Kinetic and spectroscopic data for S I67A indicate that this residue is not essential for O2 or substrate binding. The data for S167H show that the ferrous-oxy complex is dramatically destabilised, which is similar to the behaviour observed in rhTDO. The implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.;In Chapter 3, it was shown that 1-methyL-tryptophan is a substrate for rhIDO and S167A. However, no activity was observed for rhTDO. Substitution of an active site histidine residue in rhTDO (H76S) allows accommodation of the additional methyl group and 1-methyL-tryptophan turnover to occur. These observations suggest that deprotonation of the indole N 1 is not essential for catalysis, and an alternative reaction mechanism is presented. Additional experiments using EPR and 1H ENDOR spectroscopy were used to examine the surrounding environment of the heme iron. The results reveal important information on the surrounding environment of the heme-bound dioxygen and the interactions present in the ternary complex. The mechanistic implications of such interactions are discussed in this work.;In Chapter 5, we undertook site-directed mutagenesis of several active site residues and the role of each residue on dioxygen, substrate binding and in catalysis was examined. We found the conserved residue R231 plays a key role in substrate binding and is likely to do so in all heme dioxygenase enzymes. The F227A variant was found to be catalytically competent for L-tryptophan turnover and suggests that this residue is not involved in substrate recognition like previously proposed.;In Chapter 6, we have shown that rhTDO and rhIDO can utilise hydrogen peroxide as an alternative oxygen source to dioxygen. For rhTDO, approximately two equivalents of H2O2 were consumed in the production of one molecule of N-formyl-kynurenine, suggesting that an alternative mechanistic pathway is used with hydrogen peroxide

The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase catalyzes the O₂-dependent oxidation of l-tryptophan (l-Trp) to <i>N</i>-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O₂ and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O₂ binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (<i>E</i>_m⁰) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between <i>E</i>_m⁰ (that increases in the presence of l-Trp) and the rate constant for O₂ binding. This means that the initial formation of ferric superoxide (Fe³⁺–O₂^•–) from Fe²⁺-O₂ becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (<i>k</i>_cat and <i>E</i>_m⁰) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes

Probing the Ternary Complexes of Indoleamine and Tryptophan 2,3-Dioxygenases by Cryoreduction EPR and ENDOR Spectroscopy

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and <i>Shewanella oneidensis</i> (sIDO) indoleamine 2,3-dioxygenases, <i>Xanthomonas campestris</i> (<i>Xc</i>TDO) tryptophan 2,3-dioxygenase, and the H55S variant of <i>Xc</i>TDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O₂-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and ¹H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O₂, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O₂ into the C₂−C₃ double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water