Structure of the Plant Alternative Oxidase

Abstract

All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool. In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe. Mitochondrial respiratory analysis reveals that S. pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity. Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center. We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively. These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX. The alternative oxidase (AOX)1 is a ubiquinol:oxygen oxidoreductase that catalyzes the four electron reduction of oxygen to water (for recent reviews, see Refs. 1-4). This terminal oxidase branches from the cytochrome pathway at the level of the ubiquinone pool and is non-protonmotive. The AOX is resistant to inhibitors of the cytochrome pathway such as cyanide and antimycin A but inhibited by a number of compounds including hydroxamic acids (e.g. salicylhydroxamic acid) and n-alkyl-gallates.Two structural models of the AOX currently exist. The first model proposed by Siedow et al. (5,6) was based on relatively few AOX sequences and classified the AOX as a member of the di-iron family of proteins that also includes the R2 subunit of ribonucleotide reductase and the hydroxylase component of methane monooxygenase. Based on hydropathy analysis, the AOX was predicted to contain two transmembrane helices that are connected by a helix located in the intermembrane space (6) (Fig. 1A). Since this model was proposed, further AOX sequences were identified, resulting in the proposal by Andersson and Nordlund (7) of an alternative structural model (Fig. 1B). Although this second model also classifies the AOX as a di-iron protein, it differs in the precise ligation sphere of the di-iron center (7). For instance, one of the C-terminal Glu-X-X-His motifs identified by Siedow et al. (5,6), containing Glu-270, appeared not to be fully conserved in the newly identified sequences and consequently seemed unlikely to play a role in ligating iron. Instead, Andersson and Nordlund used a third Glu-X-X-His motif (that contains Glu-217, which is located in the intermembrane space according to the Siedow et al. model) to coordinate the iron atoms. Since such a choice implies that the transmembrane helices can no longer be retained, Andersson and Nordlund (7) proposed that the AOX is an interfacial rather than a transmembrane protein.Recently, the IMMUTANS (...

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