Structure of the trypanosome cyanide-insensitive alternative oxidase

Abstract

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O 2 reduction. diiron protein | neglected tropical diseases | monotopic membrane protein | drug target | ubiquinol oxidase

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

“…-The alkane hydroxylase AlkB, that removes alkylation damage to single stranded DNA, by efficiently demethylating 1-methyl adenines and 3-methyl cytosines [131][132][133]. -Several membrane bound enzymes [134], such as the alternative oxidase (AOX), a membrane bound enzyme isolated from plants, fungi and some parasites, involved in the respiratory chain [135][136][137], the plastid terminal oxidase (PTOX), which is involved in carotenoids biosynthesis [138], and the MME hydroxylase [139].…”

Thiol switches in mitochondria: operation and physiological relevance

“…In the active form reduced coenzyme Q10 [CoQ 10 (red)] from the electron transport chain is oxidized [CoQ 10 (ox)] and its electrons are passed to oxygen, which is eventually reduced to water by the active sites. The structure model shown has been generated based on the crystal structure of Trypanosoma brucei AOX (PDB 3VV9) (Shiba et al, 2013) using the sequence of Arabidopsis thaliana AOX1a. The N-termini are highly flexible and T. brucei AOX does not contain the cysteine residue.…”

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

“…The competing quinol oxidase (AOX) can only react with quinol from the pool. Image created from the structures of the ovine respirasome (Letts et al, 2016) and T. brucei AOX (Shiba et al, 2013). (B) Flow diagram showing flux through the CIII/CIV and AOX pathways if quinone is channeled (blue pathway) or behaves as a shared pool (red pathways).…”