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Plant Molecular Biology 53: 865–876, 2003. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 865 Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase Allison E. McDonald, Sasan Amirsadeghi and Greg C. Vanlerberghe∗ Department of Life Sciences and Department of Botany, University of Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario, M1C 1A4 Canada (∗ author for correspondence; e-mail [email protected]) Received 14 October 2003; accepted in revised form 4 December 2003 Key words: alternative oxidase, chlororespiration, endosymbiosis, orthologues, plastoquinol terminal oxidase, prokaryotic Abstract The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an α-proteobacterium. We used PCR, RTPCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins. Abbreviations: AOX, alternative oxidase; PTOX, plastoquinol (formerly plastid) terminal oxidase Introduction The mitochondrial electron transport chain in plants as well as some algae, fungi and protists is branched so that electrons at ubiquinol can be transferred to oxygen via the familiar cytochrome path or they may be transferred directly to oxygen via a ubiquinol oxidase called alternative oxidase (AOX; Berthold et al., 2000; Siedow and Umbach, 2000). Potential metabolic roles for this oxidase in plants have been recently reviewed and include a means to stabilize the reduction state of electron transport chain components, thereby controlling the mitochondrial generation of re- active oxygen species (Simons and Lambers, 1999; Møller, 2001). The presence of AOX in plants may have wide implications for growth, stress tolerance or susceptibility to programmed cell death (Moore et al., 2002; Robson and Vanlerberghe, 2002). Analysis of an Arabidopsis thaliana mutant (IMMUTANS) defective in chloroplast biogenesis led to the discovery of a plastid-localized protein with some sequence similarity to AOX (Carol et al., 1999; Wu et al., 1999). This protein, termed plastid terminal oxidase (PTOX), is localized to the thylakoid membrane and functions as a plastoquinol oxidase (Carol et al., 1999; Cournac et al, 2000; Joet et al., 2002; 866 Josse et al., 2003). The IMMUTANS mutation indicates that PTOX is required for carotenoid biosynthesis (Carol et al., 1999; Wu et al., 1999) and, more generally, it may represent the elusive terminal oxidase in chlororespiration (Peltier and Cournac, 2002). PTOX has been identified in plants and some green algae. AOX and PTOX are both members of the diiron carboxylate group of proteins. Such proteins can be distinguished by six conserved amino acids proposed to represent iron-binding residues (Berthold and Stenmark, 2003). Electron paramagnetic resonance data indicates that AOX does indeed contain a coupled binuclear iron center (Berthold et al., 2002). Both AOX and PTOX are proposed to bind interfacially to the membrane bilayer (Berthold and Stenmark, 2003). Given the endosymbiotic origin of plastids, it was previously suggested that an oxidase responsible for chlororespiration may have originated from a cyanobacterial ancestor (Scherer, 1990). Interestingly, heterologous expression experiments have shown that both plant AOX and PTOX proteins are functional in bacterial membranes (Berthold et al., 2002; Josse et al., 2003). Here, we report on orthologues of PTOX in the sequenced genomes of four different cyanobacteria and of an AOX orthologue in an αproteobacterium. This work complements and extends upon other recent reports of the appearance of these proteins in sequence databases (Finnegan et al., 2003; Stenmark and Nordlund, 2003). Materials and methods Organism and growth conditions Anabaena variabilis sp. strain PCC 7120 was from the University of Toronto Culture Collection. Cells were grown at 28–30 ◦ C in BG-11 medium (Rippka et al., 1979) and at a light intensity of ca. 50 µmol photons m−2 s−1 PAR. In silico analyses PTOX protein sequences were retrieved by tBLASTn searches at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov), the Institute for Genomic Research (TIGR) (http://www. tigr.org/tdb/), the Department of Energy Joint Genome Institute Microbial Genome Project (http://www.jgi. doe.gov/JGI_microbial/html/index.html), and Cyano Base (http://www.kazusa.or.jp/cyanobase/) with the A. thaliana PTOX protein sequence (NM_118352). A Chlamydomonas reinhardtii PTOX sequence identified in this search was then used to search the genomes available on the NCBI and CyanoBase servers and identified a PTOX protein in the genome of the cyanobacterium Anabaena variabilis PCC 7120. This sequence was then used to recover other prokaryotic PTOXs. Sequence alignment and phylogenetic analyses Nucleotide sequences were translated into amino acid sequences with the Translate tool located on the ExPASy server (http://us.expasy.org/tools/dna.html). All deduced amino acid sequences were aligned by means of Clustal X Version 1.81 with default gap penalties (Thompson et al., 1997). Only sequences complete enough to include all four of the iron-binding motifs (Andersson and Nordlund, 1999; Berthold et al., 2002) were used in multiple sequence alignments and for the generation of phylogenies. The first and fourth iron-binding motifs served as the boundaries for analysis. This region encompasses a total of ca. 146 amino acids for AOX proteins, and ca. 165 amino acids for PTOX proteins. In total, 67 proteins (52 AOX, 15 PTOX) in gene databases were complete enough for these analyses. Species names and accession numbers for these can be found in Figure 4. Phylogenetic analyses were conducted with MEGA version 2.1 (Kumar et al., 2001). The neighborjoining method was used with the p-distance model, but phylogenies generated with the number of distances model or the Poisson model yielded identical topologies. Bootstrap analyses consisted of 1000 replicates. Phylogenies were also generated using the maximum parsimony method in order to verify topology. Phylogenies were prepared for publication with Adobe Illustrator 9.0 (Adobe Systems, California). DNA and RNA analyses DNA and RNA were isolated from A. variabilis with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). DNA was eliminated from RNA samples prior to RT-PCR with amplification grade DNase I (Invitrogen Life Technologies). Oligonucleotide primers for PCR and RT-PCR were designed with Omiga 2.0 software (Genetics Computer Group, Madison, WI). Two primers were designed to amplify a 511 bp PTOX fragment from A. variabilis (forward, 5 -TCCTCGCTTTTATGTACTAGAAACC-3; reverse, 5 -GTGTTCCATTTCATCATCACG-3). 867 PCR was conducted in a reaction containing 5 µl 10× PCR buffer, 1 µl 10 mM dNTP mixture, 2 µl of each primer (10 µM), 2 µl DNA (0.3 µg/µl) 0.25 µl Taq DNA polymerase, and 38 µl dH2 O, all covered by 2 drops of mineral oil. The thermal cycler program consisted of 2 min at 94 ◦ C, followed by 36 cycles of 45 s at 94 ◦ C, 30 s at 49 ◦ C, and 2 min at 58 ◦ C. A final 10 min extension at 58 ◦ C was performed before cooling the tubes to 4 ◦ C. Aliquots of the PCR reaction were then analyzed by restriction digests and agarose gel electrophoresis. RT-PCR was performed with the Access RT-PCR System (Promega, Madison, WI). The reaction contained 1 µg RNA, 1 µl primers (50 pmol), 1 µl AMV reverse transcriptase, 1 µl Tfl DNA polymerase, 10 µl AMV/Tfl 5× buffer, 1 µl dNTP mix (10 mM), 2 µl of 25 mM MgSO4 and DEPC-treated water up to 50 µl. The thermal conditions used were: first-strand cDNA synthesis, 48 ◦ C for 45 min (1 cycle); reverse transcriptase inactivation and initial denaturation, 94 ◦ C for 2 min (1 cycle); denaturation, 94 ◦ C for 30 s; annealing and extension, 60 ◦ C for 1 min followed by 68 ◦ C for 2 min (40 cycles); final extension, 68 ◦ C for 7 min (1 cycle). An aliquot of the RT-PCR reaction was then analyzed by agarose gel electrophoresis. For northern analyses, total cellular RNA (20 µg) was separated on agarose gels containing formaldehyde and transferred onto Hybond-N membrane (Amersham Biosciences) with standard protocols (Maniatis et al., 1982). An RNA ladder was included on the gel. A 511 bp fragment of A. variabilis PTOX cDNA amplified by RT-PCR (see above) and radiolabeled with the T7 Quick Prime Kit (Amersham Biosciences) was used as the probe. Hybridization and wash conditions were done according to Church and Gilbert (1984) followed by exposure to X-ray film. Results Identification of eukaryotic and prokaryotic AOX and PTOX proteins Using the strategies outlined in Materials and methods, we sought to identify all AOX and PTOX proteins present in the public sequence databases. After distinguishing in our search results between PTOX and AOX proteins (see below), 20 PTOX sequences were found, including the five sequences previously annotated for the plants A. thaliana, Capsicum annuum, Gossypium hirsutum, Lycopersicon esculentum and Figure 1. Identification and expression of PTOX in the cyanobacterium Anabaena variabilis. A. Analysis by agarose gel electrophoresis and restriction digests of PCR reaction products generated with DNA isolated from Anabaena variabilis PCC 7120 and the designed primers. Lanes: 1, DNA ladder; 2, 511 bp PCR product; 3, PCR product digested with ClaI (460 and 51 bp fragments); 4, PCR product digested with PvuII (278 and 213 bp fragments); 5, PCR product digested with ClaI and PvuII (227 and 213 bp fragments); 6, control PCR without template DNA; 7, control PCR reaction without Taq DNA polymerase; 8, DNA ladder. B. Analysis by agarose gel electrophoresis of RT-PCR reaction products generated with RNA isolated from A. variabilis PCC 7120 and the designed primers. Lanes: 1, DNA ladder; 2, RT-PCR kit control 323 bp product; 3–7, RT-PCR reaction products (3 and 5, template RNA was not DNase-treated prior to RT-PCR; 5 and 6, RT-PCR reaction contained no reverse transcriptase; 7, RT-PCR reaction contained no template RNA). The results show that a reaction product of the expected size (511 bp) is generated with DNase-treated template RNA in the presence (lane 4) but not the absence (lane 6) of reverse transcriptase, thus precluding the possibility that the product is being amplified from contaminating DNA in the template RNA. C. Northern blot analysis of total RNA showing the expression of A. variabilis PTOX. The left panel shows the ethidium bromide-stained gel. The right panel shows the northern blot. An RNA ladder predicted the transcript to be ca. 800 bp. 868 Table 1. Identification of PTOX proteins in cyanobacteria, algae and higher plants. Group Species Accession number or identifier Previous annotation Anabaena variabilis PCC 7120 Gloeobacter violaceus PCC 7421 Prochlorococcus marinus MED4 Synechococcus sp. WH 8102 NP_486136a gll0601c CAE18795a NP_896980a oxidase oxidase hypothetical protein hypothetical protein Bigelowiella natans Chlamydomonas reinhardtii AY267664a AAM12876a chloroplast quinol-to-oxygen oxidoreductase chloroplast quinol-to-oxygen oxidoreductase Hordeum vulgare Oryza sativa Secale cereale Sorghum bicolor Triticum aestivum Zea mays TC88984b AAC35554a BE704987a TC41730b TC68206b TC163623b EST of unknown function oxidase EST of unknown function oxidase oxidase oxidase Arabidopsis thaliana Capsicum annuum Glycine max Gossypium hirsutum Lotus japonicus Lycopersicon esculentum Medicago truncatula Mesembryanthemum crystallinum AF098072a AF177981a BG315967a AI730398a BI419452a AF302932a TC80949b TC3799b IMMUTANS / PTOX PTOX hypothetical 38.7 kDa protein PTOX EST of unknown function GHOST / PTOX EST of unknown function PTOX Cyanobacteria Algae Monocotyledons Eudicotyledons a at the GenBank database; b at the TIGR database; c at CyanoBase. Mesembryanthemum crystallinum (Table 1). In the Oryza sativa, Sorghum bicolor, Triticum aestivum and Zea mays plants, proteins previously annotated simply as oxidases can now also be classified as PTOXs. In addition, several ESTs of unknown function and several hypothetical proteins in the plants Glycine max, Hordeum vulgare, Lotus japonicus, Medicago truncatula and Secale cereale are also PTOXs. A protein in the green alga Chlamydomonas reinhardtii and one in the alga Bigelowiella natans characterized as chloroplast quinol-to-oxygen oxidoreductases can now also be classified as PTOXs. Our search also recovered many AOX proteins from a wide variety of taxa including plants, algae, oomycetes, fungi, and protists. Significantly, our database searches also revealed prokaryotic orthologues of AOX and PTOX proteins. PTOXs were found in the sequenced genomes of four cyanobacteria: Anabaena variabilis PCC 7120, Synechococcus sp. WH 8102, Prochlorococcus marinus MED4, and Gloeobacter violaceus PCC 7421 (Table 1), while an AOX was retrieved from the genome of the α-proteobacterium Novosphigobium aromaticivorans. The presence of these prokaryotic proteins in the database (with the exception of that from G. violaceus) has been recently reported (Finnegan et al., 2003; Stenmark and Nordlund, 2003). Expression of the PTOX orthologue in cyanobacteria To confirm the presence and expression of a PTOX orthologue in cyanobacteria, we designed primers to amplify a PTOX fragment from A. variabilis PCC 7120. PCR of DNA was used to confirm the presence of PTOX in the prokaryotic genome. Analysis of the PCR reaction indicated a single product of the expected size (511 bp) and restriction digests of this PCR product generated, in each case, fragments of the expected size (Figure 1A). RT-PCR of DNase-treated RNA extracted from A. variabilis was used to confirm expression of the PTOX orthologue. RT-PCR (with the same primers as 869 above) again generated the expected 511 bp product, confirming the presence of PTOX transcript (Figure 1B). Expression was also confirmed by northern analysis of total RNA, which generated a band of ca. 800 bp (Figure 1C). The A. variabilis PTOX sequence was analyzed in the upstream region of the start codon with Softberry’s BPROM software (http://www.softberry.com). This analysis predicted a Pribnow box (GTTTATAAC) at position –60 to –52 (10 nucleotides from where the actual transcription begins) and a –35 region (CTCACA) at position –83 to –78 relative to the PTOX start codon. Furthermore, secondary structure in the untranslated region 200 bp downstream of the stop codon was analyzed with the RNA Folding application of the mfold server (http://www.bioinfo.rpi.edu/applications/mfold/). This analysis revealed a hairpin loop at position 50–67 relative to the PTOX stop codon with a closing pair A and U at positions 54 and 63. The predicted hairpin loop might serve as a transcription terminator structure. Considering these findings, a transcript size of about 815 bp is predicted, which is in good agreement with the size of transcript observed (Figure 1C). Multiple sequence alignment of AOX and PTOX proteins We examined the level of sequence similarity between AOX and PTOX proteins with a large set of sequences that included many newly found eukaryotic proteins and, most importantly, the recently found prokaryotic proteins. Both AOX and PTOX are thought to contain a diiron carboxylate centre in their active site that is coordinated by two conserved His residues and four other carboxylate (Glu) residues, all comprising four iron-binding motifs distributed within the central region of the protein sequence (Andersson and Nordlund, 1999; Berthold et al., 2000, 2002). Hence, we focused our analysis on the protein sequence bordered by the first and fourth iron-binding motifs, hypothesizing that a high degree of sequence conservation would exist in this central region to maintain protein function but that there might nonetheless exist differences between the distantly related AOX and PTOX groups (Carol and Kuntz, 2001) that could be used to differentiate the two groups. In all, 52 AOX and 15 PTOX sequences found in public databases encompassed all four iron-binding motifs and a multiple sequence alignment of these revealed consistent similarities and differences between AOX and PTOX proteins. To illustrate this, a smaller alignment including just eight AOX and six PTOX sequences is shown in Figure 2. This alignment includes thirteen species from diverse taxa and is representative of the diversity in AOX and PTOX protein sequences seen in the larger alignment. The alignment showed that all six putative ironbinding residues (E-183, E-222, H-225, E-273, E-324, H-327) are conserved in all PTOX and AOX proteins (Figure 2). Besides these, the following amino acids were also completely conserved: L-182, A-186, H198, N-221, A-276, Y-280, and D-323 (numbering according to Berthold et al., 2000). The alignment also showed the presence of a ca. 20 amino acid insert between the third and fourth iron-binding motifs of all eukaryotic PTOX proteins that all AOX proteins lacked (Carol et al., 1999). Interestingly, the insert was also present in the PTOX of the cyanobacteria A. variabilis and G. violaceus but was absent in P. marinus and reduced to about half the length in Synechococcus (Figure 2). Toward the N-terminus, there are significant differences between the PTOX proteins of plants, algae and cyanobacteria (data not shown). The N-terminus of the C. reinhardtii PTOX is extremely long compared to all others with 278 amino acid residues prior to iron-binding motif 1. Conversely, the N-termini of the cyanobacterial PTOXs are very short: 27 amino acids in A. variabilis and G. violaceus, 30 in P. marinus and 36 in Synechococcus sp. The length of the A. thaliana PTOX N-terminus is between those of algae and cyanobacteria at 134 amino acids, for which the first 51 amino acids comprise the plastid targeting peptide (Carol and Kuntz, 2001). Similarly, the N-terminus of the N. aromaticivorans AOX is extremely short compared to AOXs of plants, algae, and fungi (data not shown). While the length of the N-terminus of the C. reinhardti AOX1, A. thaliana AOX2, and N. crassa AOX is 187, 179 and 150 amino acids, respectively, that of N. aromaticivorans is only 47 amino acids prior to iron-binding motif 1. The multiple sequence alignment showed that consistent differences existed between the iron-binding motifs of the 52 AOX proteins compared to the 15 PTOX proteins. For example, in iron-binding motif 1, the sixth amino acid is an A or G in AOX, but an R in PTOX, while the ninth amino acid is a G in AOX, but a Y in PTOX (Figure 3). In a similar way, consistent differences can be established for each of the other iron-binding motifs (Figure 3). Of special note is the third amino acid in iron-binding motif 4. It is usually an A in AOX and a D in PTOX. This residue 870 Figure 2. A multiple sequence alignment of eight AOX and six PTOX proteins. This alignment includes proteins from thirteen species representative of a wide range of prokaryotic and eukaryotic taxa. Stars above the alignment indicate identical amino acids in all sequences analyzed. These include two His and four Glu resides that are putative iron-binding residues (see Figure 3). The colors and the other symbols above the alignment follow the default conventions defined by the ClustalX program, regarding conservation of amino acids between sequences. The PTOX insertion refers to a 19–20 amino acid insert that is present in most PTOX proteins but lacking in AOX proteins. The numbering of important residues (see Discussion) is the same as in Berthold et al. (2000) and represents the A. thaliana Aox1a sequence (AF370166). is critical for antibody recognition by a widely used monoclonal antibody to AOX termed AOA (Elthon et al., 1989). Substitution of the third amino acid (A) abolishes the ability of AOA to recognize AOX (Finnegan et al., 1999). Clearly, this residue differs in PTOX, providing an explanation for the apparent inability of the AOA antibody to recognize PTOX proteins. Based on this logic, we hypothesize that the AOA antibody would also fail to recognize an AOX protein of Zea mays (AAL27795) and the AOX of D. discoideum (BAB82989). The differences in iron-binding motifs (Figure 3) hold true for AOX and PTOX proteins from a wide range of prokaryotic and eukaryotic taxa and, together, should facilitate the annotation of new proteins in sequence databases. members. The two algal PTOXs group together, as do the PTOXs from the cyanobacteria A. variabilis and G. violaceus. These two cyanobacterial PTOXs do not group with the other cyanobacterial proteins, but rather are placed between the plant and algal groups. This placement of A. variabilis and G. vi- Global phylogeny of prokaryotic and eukaryotic AOX and PTOX proteins The 52 AOX and 15 PTOX sequences were used to generate an unrooted protein phylogeny by means of distance methods. This analysis showed that the prokaryotic and eukaryotic PTOXs grouped separate from the eukaryotic and prokaryotic AOXs (Figure 4). Within the PTOX group, the proteins from Synechococcus sp.and P. marinus are the most divergent Figure 3. Comparison of the iron-binding motifs of AOX and PTOX proteins. Black circles show the putative iron-binding residues, all of which are completely conserved between AOX and PTOX proteins. Black arrows indicate amino acids that can be reliably used to distinguish between AOX and PTOX proteins. The star indicates a key residue required for recognition by the AOX antibody AOA (Elthon et al., 1989; Finnegan et al., 1999). The first residue in iron-binding motif 1 corresponds to the first amino acid in the Figure 2 alignment. Results are based upon the analysis of 52 AOX and 15 PTOX sequences available in public databases. 871 olaceus was also confirmed by maximum parsimony analysis. Amongst plants, the monocot PTOXs are separate from the dicot PTOXs. Within the AOX group, the proteins from the slime mold D. discoideum and the animal parasite T. brucei brucei are the most divergent members (100% bootstrap support). The remainder of AOX proteins fall into two large groups, one containing plants and the α-proteobacterial protein, the other containing fungi, green algae, and oomycetes. Several plant species contain a small AOX gene family and so in some cases the phylogeny includes multiple proteins from one species. Consistent with previous results, the plant proteins fall into two subgroups (98% bootstrap support), one found exclusively in dicots, the other found in both monocots and dicots (Considine et al., 2002). Within the fungi, the AOX of Cryptococcus neoformans is the most divergent. The remaining fungal AOXs fall into two subfamilies (81% bootstrap support). As with plants, there is evidence for the expansion of fungal AOXs, as Candida albicans has two AOX proteins. Discussion This work explores the presence of prokaryotic orthologues of plastid PTOX and mitochondrial AOX proteins. PTOX proteins are found in the sequenced genomes of four different cyanobacteria (three of which were recently reported by Finnegan et al., 2003) while an AOX protein is present in the α-proteobacterium N. aromaticivorans as recently reported by Stenmark and Nordlund (2003). PCR confirmed the presence of PTOX in the genome of the cyanobacterium A. variabilis PCC 7120 while RT-PCR and northern analyses showed that the gene is indeed expressed. AOX and PTOX are both members of the membrane-bound diiron carboxylate oxidases (Berthold and Stenmark, 2003). A pairwise alignment of the AOX1a (AF370166) and PTOX proteins of A. thaliana indicates that the proteins share less than 50% sequence similarity (data not shown) and our phylogeny (as well as those of Finnegan et al., 2003) indicate that AOX and PTOX proteins represent two distinct groups. These findings support the practice that these proteins retain different common names. However, with the presence of PTOX in cyanobacteria, the naming of these proteins as plastid terminal oxidases becomes problematic. For simplicity, we sug- gest retaining the acronym PTOX, but recommend it stand for plastoquinol terminal oxidase. This designation is supported by the finding that the A. thaliana PTOX does indeed oxidize plastoquinol (Joet et al., 2002; Josse et al., 2003). Sequence conservation amongst eukaryotic and prokaryotic AOX and PTOX proteins Our multiple sequence alignment identified several residues that were conserved in all of the large set of eukaryotic and prokaryotic AOX and PTOX proteins analyzed. All the putative iron-binding residues were conserved, strongly supporting the view that these are the critical residues involved in the formation of a diiron carboxylate center in both AOX and PTOX (Berthold et al., 2000). Strict conservation amongst other residues provides a starting point toward studying various aspects of structure and function (Berthold et al., 2000). In the third iron-binding motif, the only Glu conserved in all PTOX and AOX proteins is E-273, the proposed iron-binding Glu in recent AOX models (Berthold et al., 2000). However, mutation of E-275 to N results in an inactive AOX (Albury et al., 2002). The most likely explanation for this is that even disruption of non-iron-binding residues in this region can disrupt the diiron center. Several PTOXs have an H-275, while several AOXs and PTOXs possess multiple Glu residues in the range of 273 to 275 (Figure 3). Hence, species-specific flexibility in the ligation sphere in this region is also possible (Finnegan et al., 2003). Many other conserved residues are next to, or in close proximity to, the proposed iron-binding residues. This is the case for L-182, A-186, N-221, A-276, and D-323. With the exception of A-186, the conservation of these residues has been noted before amongst a smaller group of non-prokaryotic AOX and PTOX proteins (Berthold et al., 2000). It is not known whether the conservation of these residues is simply related to their proximity to iron-binding residues, or whether they play a more direct role in catalysis, regulation, or structure. Other residues that are conserved in all of the AOX and PTOX proteins include H-198 and Y-280. Y-280 has been hypothesized to play a role in Tyr radical formation in the oxidation reaction, electron transfer, or quinol substrate interactions (Berthold et al., 2000) and recent site-directed mutagenesis of this residue to F yielded an inactive AOX (Albury et al., 2002). Alternatively, Y-258 is not conserved in all AOX and 872 Figure 4. Phylogeny of eukaryotic and prokaryotic AOX and PTOX proteins, generated by the Neighbour-Joining method with the p-distance model. Bootstrap values are based on 1000 trials. Branch lengths are indicated by the scale at the bottom left. 873 PTOX proteins (it is a T in Synechococcus sp.) suggesting it is not a critical Tyr for function. Consistent with this hypothesis, the mutation of this residue has no effect on AOX activity (Albury et al., 2002). Including the iron-binding residues, a total of 13 amino acids are completely conserved in the region bordered by the first and fourth iron-binding motifs of all AOX and PTOX proteins. To our knowledge, the following conserved residues have not yet been investigated: L-182, E-183, A-186, H-198, N-221, A-276, and D-323. The endosymbiotic origin of eukaryotic AOX and PTOX proteins The phylogeny of eukaryotic and prokaryotic AOX and PTOX proteins indicates that AOX and PTOX proteins represent two distinct groups. Nonetheless, given their sequence similarity, we assume that these proteins would have at one time arisen from a single bacterial ancestral protein, as recently proposed by Finnegan et al. (2003). Below, we offer two scenarios for the origin of eukaryotic AOX and PTOX proteins. The prokaryotic AOX sequence is found in an α-proteobacterium while the prokaryotic PTOX sequence is found in several cyanobacteria. This is particularly intriguing given that an α-proteobacteriallike organism is thought to have given rise to mitochondria (Gray et al., 2001) while the chloroplast is thought to have arisen from a cyanobacterial symbiont (Cavalier-Smith, 2002). The most straightforward proposal, then, is that modern-day mitochondrial AOXs arose from the endosymbiotic event that gave rise to mitochondria while modern-day chloroplast PTOXs arose from the endosymbiotic event that gave rise to chloroplasts (Finnegan et al., 2003). Given this, the divergence of AOX and PTOX proteins would have begun prior to the endosymbiotic events that gave rise to eukaryotes. Our phylogeny supports this in that the prokaryotic AOX is more similar to the eukaryotic AOX than to the prokaryotic PTOX and conversely, the prokaryotic PTOX is more similar to the eukaryotic PTOX than to the prokaryotic AOX. Assuming an endosymbiotic origin for these two proteins, the genes encoding these proteins would have later moved from the organelle genomes to the nucleus or in some cases simply been lost from the organism. An alternative possibility is that an ancestral AOX or PTOX protein was introduced to the eukaryotic line through a single endosymbiotic event, upon which the ancestral gene was relocated to the nuclear genome and then duplicated. Over time, these copies would diverge to give rise to AOX and PTOX proteins being targeted to different organelles. Recently, Martin et al. (2002) compared 24 990 proteins encoded in the A. thaliana nuclear genome to the proteins from three cyanobacterial genomes and estimated that ca. 18% of the protein-coding nuclear genes (ca. 4500 genes) were acquired from the cyanobacterial ancestor of plastids. Significantly, more than half of these proteins are predicted to now be non-plastid, indicating that gene origin is not a good predictor of protein compartmentation. Based on such findings, it seems reasonable to suggest that AOX and PTOX may have both arisen from a single gene in the cyanobacterial or proto-mitochondrial endosymbiont that subsequently diverged into the two proteins after endosymbiosis. However, if this were the case, we would expect AOX and PTOX proteins to group more closely together than our protein phylogeny indicates. This model would also fail to readily explain the presence of AOX and PTOX sequences in extant organisms that are the closest living relatives to proto-mitochondria and proto-chloroplasts, respectively. The AOX proteins It is proposed that the α-protobacteria are the closest representation of the proto-mitochondrion. It is interesting that the AOX found in the α-proteobacterium is most closely related to higher-plant AOXs. For instance, one might have expected it to group more closely with the trypanosomal AOX given that organisms in this group are known to be amongst the earliest emerging mitochondrial protists (Emelyanov, 2003). This seemingly odd result may be explained if one considers that the genes encoding the plant AOXs would have originally been present in mitochondrial DNA. In extant organisms, mitochondrial DNA is classified as either ancestral or derived (Gray et al., 1999). Ancestral groups (which include higher plants) possess a gene content that approximates that of ancestral protist mitochondrial DNA, suggesting that such DNA has been buffered against extensive loss. Derived groups (which includes fungi and algae) have undergone a great deal of reductionism in their mitochondrial genomes. When viewed in this light, the clustering of the α-proteobacterial AOX with the plant AOX group is less surprising. This may also explain the clustering of the algal, oomycete and fungal AOXs. Another possibility is that the similarity of the bacterial AOX to that of plants is due to horizontal gene 874 transfer from plants to bacteria as previously suggested (Finnegan et al., 2003; Stenmark and Nordlund, 2003). However, our BLAST searches found no evidence for such transfer of AOX to other less ancient bacteria. Also, there is still much debate over the range and frequency of horizontal gene transfer from plants to bacteria (Kurland et al., 2003). It is considered extremely rare and is constrained by the bacterium needing to contain DNA of significant homology to the transfer DNA and by the recombination event surviving the action of mismatch repairing enzymes (de Vries et al., 2001; Tepfer et al., 2003). The PTOX proteins The phylogeny indicated two groups of PTOX proteins within the cyanobacteria. Synechococcus and P. marinus grouped separately from A. variabilis and G. violaceus, with the algal PTOXs between them. Interestingly, Rubisco phylogenies classify Synechococcus and P. marinus as α-cyanobacteria, while A. variabilis is a β-cyanobacterium (Badger and Price, 2003). It is believed that the divergence of these two groups was a very ancient event that may have pre-dated primary endosymbiosis. Studies also suggest that βcyanobacterial Rubisco Form 1B, such as that found in A. variabilis, is more closely related to that of higher plants than is the α-cyanobacterial Rubisco Form 1A (Badger and Price, 2003). Similarly, the PTOX phylogeny shows that plant/algal PTOXs are more closely related to the PTOXs of β- than α-cyanobacteria. PTOXs are thus far only found in photosynthetic organisms. Despite rigorous searching, no PTOX protein could be identified in fungi or non-photosynthetic heterotrophs. This is most likely the result of chloroplast loss (Cavalier-Smith, 2002). The clustering of the PTOX of the chlorarachniophyte Bigelowiella natans with that of C. reinhardtii is consistent with the theory that B. natans acquired photosynthesis secondarily from a green alga (Figure 4; Archibald et al., 2003). Interestingly, no PTOX was found in the sequenced genome of the commonly studied Synechocystis sp. PCC 6803, indicating that not all photosynthetic organisms have PTOX. Peltier and Cournac, 2002). A chlororespiration-like path is also hypothesized to exist in cyanobacteria (Berry et al., 2002; Schreiber et al., 2002) and so one working hypothesis is that PTOX plays a role in such a path. To our knowledge, terminal oxidases potentially involved in chlororespiration-like pathways in cyanobacteria have only been extensively studied in Synechocystis sp. PCC 6803, a species for which we found no evidence of a PTOX. Indeed, previous analysis of the genome sequence indicated that, in addition to the aa3 -type cyt c oxidase (CtaI), Synechocystis harbored two additional terminal respiratory oxidases (Howitt and Vermass, 1998). These were a quinol oxidase of the cyt bd type (Cyd) and an oxidase resembling another cyt aa3 -type cyt c oxidase (CtaII). Cyd (but not CtaII) can participate in plastoquinol oxidation in the thylakoids of this species (Berry et al., 2002; Berry, 2003), suggesting it to be the most functionally similar to a PTOX. Interestingly, A. variabilis contains both Cyd genes and a PTOX (Kaneko et al., 2001). The complete genomes of two different isolates of P. marinus (MIT9313 and MED4) are available but we only find PTOX in P. marinus MED4 (Table 1). Interestingly, MIT9313 is adapted to low light levels and, despite having a much larger genome than MED4, it lacks many of the high-light-inducible proteins found in MED4 (Ting et al., 2002; Rocap et al., 2003). This may indicate that PTOX is required for high-light acclimation. It has been suggested that the chlororespiration-like pathways in cyanobacteria may act to prevent over-reduction of the plastoquinone pool under high light (Berry et al., 2002). It has also been shown that PTOX is induced by high-light stress in A. thaliana (Rizhsky et al., 2002). Acknowledgments This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada and the Premiers Research Excellence Award of Ontario (both to G.C.V.) and by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (to A.E.M.). 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