Abstract
The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.
Highlights
The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists
Mutagenesis and Expression—The A. thaliana alternative oxidase gene AOX1a was amplified with polymerase chain reaction (PCR) using pAOX [22] as a template, and the primers and, and cloned into the EcoRI-BamHI sites of pMALc2 (New England BioLabs) such that the coding region of AOX was fused to the C terminus of MBP
A single gene product was shown to be sufficient to give an active AOX when it was reported that the AOX1a gene from an A. thaliana library restored aerobic respiration in a hemedeficient E. coli mutant [22]
Summary
In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. It has been suggested that here the presence of AOX serves to prevent overreduction of the quinone pool and in so doing minimizes the release of reactive oxygen species Evidence consistent with this role has been obtained in cultured tobacco cells [8]. We have mutated several putative iron ligands and show loss of this distinctive mixed-valent EPR signal, in agreement with the assignment of the EPR signal to the diiron center in the AOX
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