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 Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO 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-O(2)-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and (1)H 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 O(2), 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 O(2) into the C(2)-C(3) double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

The redox properties of ascorbate peroxidase.

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.

Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis.

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.