At the gene level, conservation among cytochrome b
561-encoding genes is not very high [7
]. The genomic organization usually comprises four or five exons in plants and mammals (Figure ). 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 b
561 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 b
561. The physiological implication of these altered cytochrome b
561 structures is unclear. Interestingly, the alternative splice products have in each case retained the cytochrome b
561 'core structure' as defined by Ponting [24
], 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 b
561-like sequences is restricted to animals and plants. Some fungal species have been demonstrated to synthesize the ascorbate analogs D
) or erythro-ascorbate (Candida albicans, Saccharomyces cerevisiae
) instead of ascorbate [33
]. However, these compounds occur at very low concentrations, and they probably have only limited importance - if any - as antioxidants [34
]. Ascorbate is apparently completely absent in prokaryotes [33
]. The absence of cytochromes b
561 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 b
561 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 b
561 proteins in a range of phylogenetically very distinct species (Figure ). 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 ). Although an ascorbate-reducible cytochrome b
561 has been demonstrated in several plant species [12
], this theoretically leaves open the possibility that other substrates may function as electron donors to the plant cytohromes b
561. 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 b
561 in A. thaliana
Plant and animal cytochromes b561 generally separate into two clusters in a phylogenetic tree (Figure ), 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 ).
Within the mammalian cluster of cytochromes b
561, the three paralogous proteins (from chromaffin granules, duodenal and 'ubiquitous') form separate, monophyletic groups. The presence of a cytochrome b
561-like protein in C. intestinalis
is interesting. Tunicates, the most primitive chordates, are considered the direct ancestors of vertebrates [36
], suggesting that the protein from C. intestinalis
might represent an ancestral form of the mammalian cytochromes b
561. Synonymous substitution rates for the cytochrome b
561 genes from C. intestinalis
and different vertebrates are rather low (1-1.5 × 10-9
/position/year, Table ), as compared to other nuclear-encoded mammalian genes (for example, 4.61 × 10-9
]). 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 ). 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 ). 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 ), suggesting that they diversified more recently in evolution, which is supported by our Li93 analysis (Table ).
561 are possibly involved in the regeneration of ascorbate through transmembrane electron transport [8
]. 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 b
561 protein family in different organelles. In humans, the chromaffin tissue cytochrome b
561 is present in the membrane of a subcellular secretory vesicle [8
], whereas the duodenal cytochrome b
561 is a plasma membrane protein [39
]. 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 b
561 isoforms is present in the plasma membrane [7
]. 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 b
561. The apparent absence of a cytochrome b
561 in the plasma membrane of algae [7
] can be taken as an indication that the primitive form of the protein was located in internal membranes.