Structure and reaction mechanism in the heme dioxygenases

Abstract

As members of the family of heme-dependent enzymes, the heme dioxygenases are differentiated by virtue of their ability to catalyze the oxidation of l-tryptophan to N-formylkynurenine, the first and rate-limiting step in tryptophan catabolism. In the past several years, there have been a number of important developments that have meant that established proposals for the reaction mechanism in the heme dioxygenases have required reassessment. This focused review presents a summary of these recent advances, written from a structural and mechanistic perspective. It attempts to present answers to some of the long-standing questions, to highlight as yet unresolved issues, and to explore the similarities and differences of other well-known catalytic heme enzymes such as the cytochromes P450, NO synthase, and peroxidases.

Scheme 1. Reaction Catalyzed by the Heme Dioxygenases

Scheme 2. Previous Literature Proposals^,, for the Reaction Mechanism for Heme Dioxygenases

Reaction of ferrous heme with O₂ and Trp leads to a ternary complex that reacts by base-catalysed abstraction to form a peroxyindole intermediate. This species was proposed to decay by either Criegee (blue) or dioxetane (red) pathways.

Figure 1

(A) Overall structure of human IDO,(17) showing the large (red) and small (blue) domains, the loop region (yellow), the heme (in green), the proximal histidine residue (His346, red), and the bound inhibitor, 4-phenylimidazole (green). (B) Active site of human IDO(17) with the heme colored red. There is one molecule of the inhibitor 4-phenylimidazole (green) bound to the heme iron in the structure and two molecules of the crystallization buffer (CHES, not shown) in the active site. Figures 1 and 2 were prepared using Pymol.(66)

Scheme 3. Variously Proposed Reaction Mechanisms for Heme Dioxygenases

(A) The base-catalysed abstraction mechanism.^, (B) An alternative to the base-catalysed mechanism, using abstraction of protons by the bound O₂.^, (C) Electrophilic addition.^,, (D) Radical addition.^,,, The majority opinion from crystallography,(17) mass spectrometry,(23) mutagenesis,(24) and computational work(32) concludes that mechanisms A and B are unlikely. It is not yet known whether addition at C³ or C² is most likely; both have been suggested.^−,,,.

Figure 2

(A) Overlay of X. campestris TDO (green(18)) and hIDO (white(17)), showing the active site residues (in parentheses for hIDO). (B) Substrate binding site in X. campestris TDO,(18) showing the substrate (orange) and the associated bonding interactions.

Figure 3

Overlay of the heme regions of X. campestris TDO (gray(18)) and PrnB (pale blue(27)), showing the different orientations of the substrate (l-Trp for TDO and 7-Cl-Trp for PrnB, both colored gray, with the Cl atom colored green). The heme groups are overlaid in red. (B) Reaction catalyzed by PrnB (compare to Scheme 1).

Scheme 4. Structures of the Various Tryptophan Analogues Mentioned in This Work

Scheme 5. Alternative Mechanistic Proposal

(A) Radical addition (instead of base-catalysed abstraction) leading to species X is followed by formation of a ferryl heme species (Compound II) and a proposed epoxide species.(38) Formation of an epoxide has also been suggested by computational work(42) (in fact, it was considered in an earlier study, but initially considered energetically unfavorable in the gas phase(32)). Electrophilic addition (Scheme 3C) could also involve epoxide formation through a similar (two-electron) mechanism. Possible ring opening of the epoxide is also indicated. (B) Proposed(42) mechanism for subsequent formation of NFK, starting from the same species X as in panel A, and also involving initial ring opening.

Scheme 6. Comparison of the Reaction Mechanisms in Various Heme Proteins