The alternative oxidase is a membrane-bound ubiquinol oxidase found in the majority of plants as well as many fungi and protists, including pathogenic organisms such as Trypanosoma brucei. It catalyzes a cyanide- and antimycin-A-resistant oxidation of ubiquinol and the reduction of oxygen to water, short-circuiting the mitochondrial electron-transport chain prior to proton translocation by complexes III and IV, thereby dramatically reducing ATP formation. In plants, it plays a key role in cellular metabolism, thermogenesis, and energy homeostasis and is generally considered to be a major stress-induced protein. We describe recent advances in our understanding of this protein's structure following the recent successful crystallization of the alternative oxidase from T. brucei. We focus on the nature of the active site and ubiquinol-binding channels and propose a mechanism for the reduction of oxygen to water based on these structural insights. We also consider the regulation of activity at the posttranslational and retrograde levels and highlight challenges for future research.

Summary 1.In central Corsica, Helicodiceros muscivorus (Schott ex. K. Koch) produces a protogynous inflorescence that resembles the anal area of a dead mammal and produces a foetid scent during the few hours after sunrise. Flies enter the floral chamber, pollinate the female florets and become trapped until the next morning, when pollen is shed from the male florets and the flies are released. 2. The exposed appendix exhibits a strong, unimodal episode of thermogenesis associated with scent production, reaching a maximum of 30 ° C at 15 ° C ambient temperature. The male florets in the floral chamber are highly thermogenic throughout the second night and generally maintain stable floret temperatures of about 24 ° C at ambient temperatures down to 13 ° C. 3. Maximum respiration rates of the appendix (0·45 µ mol CO 2 s − 1 g − 1 ) and the male florets (0·82 µ mol s − 1 g − 1 ) may be the highest recorded for plant tissue. 4. Thermogenesis of the appendix does not depend on ambient temperature, but that of the male florets increases with decreasing ambient temperature in most cases. However, the pattern of heat production by the males appears related more to time than to ambient temperature, hence the term 'pseudo-thermoregulation'. 5 . The behaviour and thoracic temperatures of flies emerging from captivity suggests that male floral warming does not enhance their activity.

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys1 and Cys2, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys1 with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys1. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys1 had been replaced by serine; however, Cys2 was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys1 and Cys2. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.