A phylogenetic study of cytochrome b561 proteins
© Verelst and Asard; licensee BioMed Central Ltd. 2003
Received: 27 January 2003
Accepted: 24 April 2003
Published: 28 May 2003
As an antioxidant and cofactor to numerous metabolic enzymes, ascorbate has an essential role in plants and animals. Cytochromes b561 constitute a class of intrinsic membrane proteins involved in ascorbate regeneration. Despite their importance in ascorbate metabolism, no evolutionary analysis has been presented so far on this newly described protein family.
Cytochromes b561 have been identified in a large number of phylogenetically distant species, but are absent in fungi and prokaryotes. Most species contain three or four cytochrome b561 paralogous proteins, and the encoding genes usually have four or five exons. At the protein level, sequence similarities are rather low between cytochromes b561 within a single species (34-45% identity), and among phylogenetically distant species (around 30% identity). However, particular structural features characterizing this protein family are well conserved in members from all species investigated. These features comprise six transmembrane helices, four strictly conserved histidine residues, probably coordinating the two heme molecules, and putative ascorbate and monodehydro-ascorbate (MDHA) substrate-binding sites. Analysis of plant cytochromes b561 shows a separation between those from monocotyledonous and dicotyledonous species in a phylogenetic tree.
All cytochromes b561 have probably evolved from a common ancestral protein before the separation of plants and animals. Their phyletic distribution mirrors the use of ascorbate as primary antioxidant, indicating their role in ascorbate homeostasis and antioxidative defense. In plants, the differentiation into four cytochrome b561 isoforms probably occurred before the separation between monocots and dicots.
Ascorbate (vitamin C) is generally known for its detoxification of damaging reactive oxygen species during aerobic metabolism and under stress conditions . Through the modulation of levels of reactive oxygen species, ascorbate is implicated in the control of cell expansion, cell division and programmed cell death . As a cofactor to numerous iron- and copper-containing oxygenases, ascorbate is also involved in the biosynthesis of essential molecules, such as the plant hormones ethylene and gibberellic acid, cell-wall glycoproteins and antimicrobial agents [3, 4]. The recent unraveling of the ascorbate biosynthetic pathway in plants [5, 6] has resulted in a renewed interest in this molecule, and in the recognition that much remains to be learned about its regulation and metabolism.
While our knowledge of ascorbate biosynthesis and catabolism is rapidly expanding, little is known about the mechanisms by which ascorbate is regenerated throughout the plant cell. Cytochromes b561 constitute a newly identified class of membrane proteins possibly implicated in replenishing ascorbate pools in plant cells, essential to maintaining the physiological functions of this important molecule .
Cytochromes b561 are intrinsic membrane proteins containing two heme molecules, and reducible by ascorbate [8, 9]. They have been suggested to function as electron transporters, shuttling electrons across membranes from ascorbate to an acceptor molecule. The one-electron oxidation product of ascorbate, monodehydro-ascorbate (MDHA) has been shown, at least in vitro, to function as an electron acceptor for mammalian and plant cytochromes b561 [8, 10, 11]. The cytochrome b561-catalyzed reduction of MDHA results in the regeneration of the fully reduced ascorbate molecule.
The presence of cytochromes b561 in plants was first demonstrated on the basis of their biochemical properties. Ascorbate-reducible cytochromes with a wavelength maximum near 561 nm and a typically high redox potential (E'0 around +140 mV) were found in purified plasma membrane fractions from various species [12, 13]. The availability of the primary sequence of the cytochrome b561 from bovine adrenal gland chromaffin cells [14, 15] has recently resulted in the identification of homologous sequences in plants [7, 16]. Putative cytochrome b561-encoding genes have now been identified in nearly all organisms for which considerable genomic sequence information is available, including invertebrates (insects, nematodes, platyhelminths, tunicates), vertebrates (mammals, amphibians) and plants (both monocots, dicots and gymnosperms) (this paper and [7, 17]). The presence of cytochromes b561 in a wide variety of species indicates the general importance of this class of proteins in eukaryotic cell physiology.
Members of the cytochrome b561 protein family are characterized by a number of structural features, likely to play an essential part in their function [7, 17, 18]. They are highly hydrophobic proteins with six transmembrane helices, four conserved His residues, possibly coordinating two heme molecules, and predicted substrate-binding sites for ascorbate and monodehydro-ascorbate (MDHA). The occurrence of cytochromes b561 in species phylogenetically remote as nematodes and mammals, tunicates and insects, or amphibians and plants, is intriguing. The strict conservation of essential structural features suggests that the mode of action and physiological function of these proteins may be very similar. We present here a further analysis of the similarities and differences between cytochromes b561 throughout the animal and plant kingdoms, at the level of genomic organization and protein structure. Also, we examine the phylogenetic relations between all cytochrome b561 proteins identified so far, to allow hypotheses to be made on the evolution of these ubiquitous proteins.
Analysis of cytochrome b561-encoding genes
Upon the completion of the Arabidopsis thaliana genome project in December 2000, it became possible to identify all genes encoding cytochromes b561 in a plant. Four putative b561 genes were identified in A. thaliana: Artb561-1 to -4 (for Arabidopsis thaliana cyt b561) . Genomic sequences possibly encoding cytochromes b561 were also identified in Oryza sativa and Craterostigma plantagineum. Most putative b561-encoding sequences from other plant species, however, are so far only represented by expressed sequence tags (ESTs).
Genomic sequences for cytochromes b561 are also known for a number of animal species, including Drosophila melanogaster, Caenorhabditis elegans, Homo sapiens (human) and Mus musculus (mouse). The genomes of human and mouse each encode three cytochromes b561: one involved in ascorbate regeneration inside chromaffin granules (Hosb561-1, Mumb561-1), one present in the duodenum (Hosb561-2, Mumb561-2) that may function as a ferric reductase in the plasma membrane of duodenal mucosa, and a third that seems to have a ubiquitous distribution in mammalian tissues, but whose physiological function is not known. This is tentatively referred to as 'ubiquitous cytochrome b561' (Hosb561-3, Mumb561-3) (H.A., unpublished work).
Alternative splicing of cytochrome b561-encoding genes is observed in D. melanogaster. Different transcripts are found for each of the two putative genes (CG1275 and CG8776). The first gene has two transcripts (Drmb561-1 and -2), each encoded by four exons. Drmb561-1 is only different from Drmb561-2 in that it has 86 extra amino acids at the amino terminus. This extra region (predicted to be cytoplasmic) is absent in all other cytochrome b561 proteins identified thus far. The second gene from D. melanogaster has three different transcripts, encoded by five (Drmb561-3) or six (Drmb561-4 and -5) exons. Again, the only difference is seen at the amino terminus of the encoded proteins. Drmb561-5 is nearly identical to Drmb561-3, with only a difference in 20 amino-terminal amino acids. The sequence of the Drmb561-4 protein corresponds to that of the other two proteins, except for the lack of 80 amino acids at the amino terminus. At the protein level, Drmb561-4 thus proves to be the shortest isoform, lacking the first transmembrane helix. All six helices are present in the other two isoforms.
Comparison of putative cytochrome b561 proteins
In addition to the conservation of the predicted transmembrane structures, four His residues suggested to bind the two heme groups are strictly conserved in all cytochrome b561 homologs. Furthermore, predicted binding sites for MDHA and ascorbate - as suggested by  - are conserved. A potential MDHA-binding motif - xYSLHSWxGx - with x being a hydrophobic amino acid in most proteins, is highly conserved. In the Anopheles gambiae (malaria mosquito) b561 protein, Angb561, the Ser is conservatively replaced by Thr. The Tyr in the consensus sequence is not conserved in the proteins from C. elegans - Caeb561-1, -2 and -3 - nor in Artb561-3, but it is present in all Artb561-3 orthologs identified in other plant species (data not shown).
The suggested ascorbate-binding site (ALLVYRVFR in the mammalian chromaffin cytochrome b561 ) is almost perfectly conserved in all mammalian proteins, but the degree of conservation is much lower in plants (Figure 3). The first three or four amino acids of this consensus sequence are generally hydrophobic, and the Tyr is found in all proteins except Artb561-4, in which it is replaced by His. The triplet codons for Tyr and His differ by only one nucleotide, suggesting that a single point mutation may be responsible for this substitution. The His at this position is found in all Artb561-4 orthologs from other plant species (data not shown), and therefore seems a consistent substitution in this isoform of plant cytochromes b561. The Arg from this motif is conservatively replaced by Lys in all available plant sequences. To the best of our knowledge the sequence requirement for ascorbate and MDHA binding has not been experimentally confirmed, and so it is not possible to evaluate whether the observed conservation is sufficient for the functional interaction with the ascorbate-MDHA redox couple.
In addition to the conserved structural features mentioned above, all cytochromes b561 show strict conservation of five Gly, two Pro, one Lys and one Gln (green in Figure 3). A number of aromatic residues are also well conserved (yellow in Figure 3). These have been suggested to take part in the electron transfer between the two heme molecules, possibly by electron tunneling .
Phylogeny of cytochromes b561
A closer examination of the plant group reveals the clustering of sequences from Lycopersicon esculentum (tomato, Lyeb561), L. hirsutum (Lyhb561), Solanum tuberosum (potato, Sotb561), C. plantagineum (resurrection plant, Crpb561), Beta vulgaris (beet, Bevb561-1), Populus tremula (poplar, Potb561), Medicago truncatula (barrel medic, Metb561-1), Oryza sativa (rice, Orsb561), Zea mays (maize, Zemb561), Hordeum vulgare (barley, Hovb561-1), Sorghum bicolor (sorghum, Sobb561), Triticum aestivum (wheat, Trab561-1) and T. monococcum (Trmb561) with the Artb561-1 protein, suggesting that these proteins are orthologs of Artb561-1 from A. thaliana. The localization of a cytochrome b561 from Pinus taeda (loblolly pine, Pitb561) within this cluster is not supported by a bootstrap value above 80% and should be interpreted with caution. Similarly, sequences from T. aestivum (Trab561-2), H. vulgare (Hovb561-2), M. truncatula (Metb561-2), Glycine max (soybean, Glmb561-2), Zinnia elegans (zinnia, Zieb561) and B. vulgaris (Bevb561-2) are clustered with Artb561-2. Orthologs of Artb561-3 have been identified in Mesembryanthemum crystallinum (ice plant, Mecb561-3), M. truncatula (Metb561-3) and Gossypium hirsutum (cotton, Gohb561), and Artb561-4 orthologs in M. truncatula (Metb561-4) and B. vulgaris (Bevb561-4).
Most of the plant cytochrome b561 sequences so far have been obtained from dicotyledonous plants, but for Artb561-1 and -2 some orthologs from monocotyledons are available. These form separate 'sub-clusters' within the clusters of Artb561-1 and -2 respectively, whereas an Artb561-1 ortholog from the gymnosperm P. taeda is more distantly related with these clusters. For Artb561-3 and -4 no orthologs have been identified in monocotyledonous plants so far. Within the animal group, all known chromaffin granule cytochromes b561 - from H. sapiens (human, Hosb561-1), M. musculus (mouse, Mumb561-1), Sus scrofa (pig, Susb561), Bos taurus (bovine, Botb561) and Ovis aries (sheep, Ovab561) are clustered together with the homolog from Xenopus laevis (African clawed frog, Xelb561). The duodenal cytochromes b561 from humans, mouse and rat (Hosb561-2, Mumb561-2 and Ranb561-2) form a separate group, as do the chromaffin granule cytochromes b561 and the third type of mammalian cytochrome b561 ('ubiquitous' cytochrome b561 from humans and mouse, Hosb561-3 and Mumb561-3). The position of the cytochrome b561 protein from Ciona intestinalis (Ciib561; sea squirt, a tunicate) is uncertain, as the bootstrap value is below 80%. The cytochrome b561 homologs identified in C. elegans and the flatworm Dugesia japonica could not reliably be associated with any of the clusters.
Li93 analysis of a selection of cytochrome b561-encoding genes
Dicot versus monocot
0.37 ± 0.09
0.43 ± 0.06
1.22 ± 0.38
2.88 ± 0.69
3.33 ± 0.45
12.63 ± 2.41
2.38 ± 1.18
Tunicate versus vertebrates
0.65 ± 0.13
0.78 ± 0.12
1.26 ± 0.45
1.21 ± 0.24
1.45 ± 0.22
Human versus mouse
Comparison of cytochromes b561 from dicotyledonous species to their orthologs from monocotyledons reveals that Ks and Ka values are generally both around 0.3 to 0.5. Knowing that both clades diversified 130 million years ago , the average rate of synonymous and nonsynonymous substitutions is calculated to be around 3 × 10-9 substitutions per position per year (Table 1). This is comparable to the synonymous evolution rate of other nuclear-encoded plant genes (5-30 × 10-9 substitutions/position/year ). Among dicotyledons (using estimated divergence times for different families, derived from Figure 2 in ) the average synonymous substitution rate is higher (12.63 ± 2.41 × 10-9 substitutions/position/year), but the nonsynonymous rate is very similar (2.38 ± 1.18 × 10-9 substitutions/position/year). For the resurrection plant (C. plantagineum), however, which has adapted to life in an extremely dry environment, a particularly high nonsynonymous substitution rate was found as compared to the ortholog from potato (13.25 × 10-9 substitutions/position/year), whereas the synonymous rate was the same as for the orthologs in other dicots (Table 1).
Ks and Ka were also calculated for animal cytochrome b561 genes. Comparing the gene from the tunicate C. intestinalis (Ciib561), which branched off the vertebrate lineage around 540 million years ago , with the chromaffin granule cytochrome b561 genes from vertebrates (X. laevis, H. sapiens and M. musculus) gives a Ks of around 0.65 and a Ka of 0.8. This is remarkably low, considering the large phyletic distance between tunicates and vertebrates, and implies synonymous and nonsynonymous substitution rates of 1-1.5 × 10-9 substitutions/position/year.
The Gu99 likelihood ratio test in the DIVERGE software package  was used to examine whether proteins from different branches in the phylogenetic tree have different functional constraints, and whether they may have functionally diverged. The Theta ML value, indicative for the level of functional divergence between proteins , is relatively low (0.213) according to , when monocotyledonous and dicotyledonous Artb561-1 orthologs are compared. This suggests that these orthologs are likely to have similar biochemical functions. The Gu99 likelihood assay also indicated that a functional adaptation may have occurred at only five amino acids between these orthologs, supporting their functional similarity. A similar analysis of Artb561-1 and -2 orthologs yielded a theta ML value of 0.533, and 22 residues that may have functionally adapted. These numbers are still rather low . However, four of these residues (A, I, S and P) are situated in the predicted ascorbate-binding site (AIISYKSLP in Artb561-1). A similar analysis for the mammalian cyts b561 (chromaffin cyts b561 versus the cluster with the other two mammalian cyts b561) resulted in a very low theta ML of 0.031, and no residues likely to have an altered function.
At the gene level, conservation among cytochrome b561-encoding genes is not very high . The genomic organization usually comprises four or five exons in plants and mammals (Figure 1). In addition to genes with four or five exons, invertebrates contain homologous genes with only two exons (C. elegans F55H2.5), or with six exons (D. melanogaster). Splice variants are observed for D. melanogaster CG1275 and CG8776. This alternative splicing results in a cytochrome b561 protein with an additional amino-terminal region (Drmb561-1), and in a protein that lacks the first of the six transmembrane helices (Drmb561-4), commonly present in plant and mammalian cytochromes b561. The physiological implication of these altered cytochrome b561 structures is unclear. Interestingly, the alternative splice products have in each case retained the cytochrome b561 'core structure' as defined by Ponting , that is, four transmembrane helices containing the conserved heme-ligating His residues and the predicted substrate-binding sites.
Despite the availability of extensive genomic sequence information from fungi and prokaryotes, the presence of cytochrome b561-like sequences is restricted to animals and plants. Some fungal species have been demonstrated to synthesize the ascorbate analogs D-arabo-ascorbate (Penicillium) or erythro-ascorbate (Candida albicans, Saccharomyces cerevisiae) instead of ascorbate [33, 34]. However, these compounds occur at very low concentrations, and they probably have only limited importance - if any - as antioxidants . Ascorbate is apparently completely absent in prokaryotes . The absence of cytochromes b561 in fungi and prokaryotes may therefore be related to the absence of L-ascorbate as a major antioxidant.
Our phylogenetic analysis supports the hypothesis that all cytochrome b561 proteins have probably evolved from a single protein, present in the common ancestor of plants and animals. This conclusion is supported by the perfect conservation of the 'core structural features' in cytochrome b561 proteins in a range of phylogenetically very distinct species (Figure 3). It should be noted that the conservation of the putative ascorbate-binding sites is high in the mammalian proteins, but considerably less in the plant sequences (Figure 3). Although an ascorbate-reducible cytochrome b561 has been demonstrated in several plant species [12, 35], this theoretically leaves open the possibility that other substrates may function as electron donors to the plant cytohromes b561. The Gu99 likelihood analysis indicates that four amino-acid residues in the ascorbate-binding site may show functional adaptation, supporting the possibility of different substrate-binding site affinities among cytochromes b561 in A. thaliana.
Plant and animal cytochromes b561 generally separate into two clusters in a phylogenetic tree (Figure 4), indicating a diversification early in evolution. The pairwise similarity between each of the A. thaliana cytochromes b561 and the animal homologs is comparable (data not shown), suggesting that they diversified within an evolutionarily short time span from a single ancestral protein, after the separation between plants and animals. Cytochrome b561 proteins from invertebrates (insects, nematode and flatworm) are not tightly linked to the plant or mammalian cluster (Figure 4).
Within the mammalian cluster of cytochromes b561, the three paralogous proteins (from chromaffin granules, duodenal and 'ubiquitous') form separate, monophyletic groups. The presence of a cytochrome b561-like protein in C. intestinalis is interesting. Tunicates, the most primitive chordates, are considered the direct ancestors of vertebrates , suggesting that the protein from C. intestinalis might represent an ancestral form of the mammalian cytochromes b561. Synonymous substitution rates for the cytochrome b561 genes from C. intestinalis and different vertebrates are rather low (1-1.5 × 10-9/position/year, Table 1), as compared to other nuclear-encoded mammalian genes (for example, 4.61 × 10-9/position/year ). Nonsynonymous substitution rates are in the same range, indicating a good conservation at the protein level.
In addition to conclusions on the evolutionary relationship between cytochromes b561 in different species, the cladogram points to interesting relations between cytochrome b561 isoforms within a single species. The four paralogous proteins from A. thaliana (Artb561-1, -2, -3 and -4) form separate clusters with their respective orthologs from other plant species (Figure 4). It is thus likely that gene duplication events had already occurred in algae or primitive plants. The identification of a cytochrome b561 in the gymnosperm P. taeda (Pitb561) supports this suggestion.
The clusters containing the A. thaliana isoforms Artb561-1 and Artb561-2 also contain homologous sequences from both dicotyledons and monocotyledons. In both cases, the proteins from monocots and dicots form separate sub-clusters. This observation suggests that the four different cytochromes b561 have evolved separately in dicots and monocots, and hence that the diversification between these proteins had already occurred in their common ancestor. Substitution rates in cytochrome b561 genes from monocots and dicots are comparable to those in other plant nuclear-encoded genes (Table 1). The Artb561-1 ortholog in the drought-resistant resurrection plant (Crpb561), seems to have a remarkably high rate of nonsynonymous substitution (13.25/position/year). Interestingly, the Artb561-2 and -4 proteins tend to group in the same cluster (Figure 4), suggesting that they diversified more recently in evolution, which is supported by our Li93 analysis (Table 1).
Cyts b561 are possibly involved in the regeneration of ascorbate through transmembrane electron transport [8, 10, 11, 18]. This functional conservation is supported by our likelihood ratio test (Gu99 test). As ascorbate is present in different subcellular organelles in plants and animals, it is not surprising to find members of the cytochrome b561 protein family in different organelles. In humans, the chromaffin tissue cytochrome b561 is present in the membrane of a subcellular secretory vesicle [8, 10, 38], whereas the duodenal cytochrome b561 is a plasma membrane protein . The subcellular localization of the third human isoform (Hosb561-3) is not yet known. In A. thaliana and several other plant species, at least one of the cytochrome b561 isoforms is present in the plasma membrane [7, 12]. The subcellular localization of the other isoforms remains to be determined. The association with different membranes raises the question on the subcellular localization of the ancestral cytochrome b561. The apparent absence of a cytochrome b561 in the plasma membrane of algae  can be taken as an indication that the primitive form of the protein was located in internal membranes.
Materials and methods
Database searches used BLAST  on the website of the National Center for Biotechnology Information (NCBI ). Multiple sequence alignments were made with Clustal W , and confirmed with POA  and T-Coffee , which have been reported to give more accurate alignments . These data were converted into a cladogram using Treecon software . Distances were calculated with Poisson correction, and for tree topology, complete linkage clustering was used. The Li93 analysis was performed with DAMBE , after aligning the genes pairwise with Clustal W and T-Coffee. The maximum likelihood analysis (Gu99) was performed with the DIVERGE software , using the phylogenetic tree from Figure 4. DIVERGE calculates a theta ML value indicative of the level of functional divergence between proteins in different clusters of the tree, and a posterior probability to trace the amino-acid positions that are likely to be responsible for the functional divergence between proteins in both clusters. Transmembrane structures were predicted with TMHMM .
The authors thank Etsuko Moriyama (UNL) for valuable advice and for critically reading the manuscript. This research was supported by grants from the Fund for Scientific Research (FWO) and the University of Antwerp; W.V. is Research Assistant of the Fund for Scientific Research - Flanders (Belgium) (FWO). A contribution of the University of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal Series No. 14061.
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