* Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Biologie Structurale et Microbiologie, Marseille Cedex, France
Laboratoire de Dynamique, Evolution et Expression des Génomes de Micro-Organismes, Université Louis-Pasteur, Strasbourg Cedex, France
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Abstract |
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Key Words: arsenite bioenergetics evolution phylogeny Rieske protein molybdopterin protein
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Introduction |
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Is arsenite oxidase from Alcaligenes faecalis therefore a relatively recent evolutionary invention built by associating redox subunits from existing electron transfer complexes into a new enzyme with a detoxifying activity? In order to detect potential further members of a putative arsenite oxidase family, we have performed a survey of fully sequenced genomes accessible in the databases. This search came up with several further examples, both bacterial and archaeal, of enzymes almost certainly belonging to this new family. A phylogenetic analysis of both subunits argues for an ancient evolutionary origin of the arsenite oxidase family. The phylogenetic relationship of its Rieske subunit to that of the Rieske/cytb complexes was analyzed and the results are discussed in the light of the evolutionary histories of both enzymes.
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Materials and Methods |
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Bacterial cultures and sample preparation
Chloroflexus aurantiacus was grown photosynthetically and membrane fragments were prepared as described previously (van Vliet, Nitschke, and Rutherford 1991; Brugna 1997). Oriented membrane samples were obtained by partial dehydration as described by Rutherford and Sétif (1990).
EPR experiments
EPR spectra were recorded on a Bruker ESP300e X-band spectrometer fitted with an Oxford Instruments liquid helium cryostat (temperature, 20°K; microwave frequency, 9.42 GHz; microwave power, 6.7 mW; modulation amplitude, 1.6 mT).
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Results |
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Biophysical Characterization of the Arsenite Oxidase from Chloroflexus aurantiacus
The electron paramagnetic resonance (EPR) spectrum of a Rieske iron sulfur cluster in membranes from phototrophically grown Chloroflexus aurantiacus was first reported almost 20 years ago (Zannoni and Ingledew 1985), and this observation was subsequently confirmed by several groups (Wynn et al. 1987; Brugna 1997). Notwithstanding its low concentration as compared with all cytochromes observable in the same samples (Wynn et al. 1987; Brugna 1997), the Chloroflexus Rieske protein has been considered to represent the respective subunit of a Rieske/cytb complex. Despite sustained efforts, however, no enzyme corresponding to an integral Rieske/cytb complex could be purified (Brugna 1997; Yanyushin 2002). The failure to isolate this enzyme is rationalized by the results of the Chloroflexus genome sequencing project, which shows that the operon encoding a typical Rieske/cytb complex is not present in the Chloroflexus genome. A search of the genome for Rieske proteinrelated genes yielded the respective subunit from arsenite oxidase as the one and only significant hit. Arsenite oxidase in Chloroflexus has thus unwittingly been studied by several groups for almost two decades. The respective results are summarized in the following:
Multiple Sequence Alignments and Phylogenetic Analyses
Figure 1 shows multiple sequence alignments of the catalytic large and the Rieske subunits. Included in the multiple alignments are selected sequences from the homologous proteins contained in DMSO-reductase, assimilatory nitrate reductase, formate dehydrogenase (fig. 1a) as well as Rieske subunits from Rieske/cytb-type enzymes (fig. 1b). The alignment of the Rieske proteins relies on exploiting the available three-dimensional coordinates for a structure-guided superposition of structurally conserved features in the N-terminal half of the protein, which is characterized by low conservation of primary sequence (a detailed description of this method will be published elsewhere). Residues involved in cofactor binding as well as the twin arginine translocation (tat) signal sequence and the subsequent hydrophobic stretch in the case of the Rieske protein are highlighted in the multiple alignments.
The obtained multiple sequence alignments (including further representatives of the various groups as indicated in the legend of figure 2) were used to create phylogenetic trees based on neighbor-joining and parsimony methods. Resulting phylogenies are shown as unrooted trees in figure 2a and b for the molybdopterin and the Rieske subunits, respectively.
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Discussion |
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As judged by the replacement of a cysteine by a serine residue in the respective terminal sequence motif (fig. 1a), the cubane cluster in all arsenite oxidases is most probably a [3Fe-4S] cluster rather than a [4Fe-4S] center as is the case in nitrate reductase and formate dehydrogenase (Jormakka et al. 2002). This is in line with the relatively high redox midpoint potential of this cluster as required by function (discussed above) and as measured in Chloroflexus (+60 mV). Two of the four residues suggested to be involved in arsenite binding (Ellis et al. 2001) are also fully conserved (fig. 1a). A further feature is the absence of the cysteine/selenocysteine residue covalently binding the molybdenum atom in nitrate reductases and formate dehydrogenases. The lack of this covalent bond in arsenite oxidases has been proposed to play a crucial role in the As tolerance of the molybdenum center in this specific enzyme (Ellis et al. 2001).
Figure 1b shows the multiple sequence alignment of Rieske proteins from arsenite oxidases and from Rieske/cytb complexes. Structurally conserved regions comprise the transmembrane helix, the ß-loop structure (Lebrun et al., unpublished data), and the cluster binding C-terminal domain. It is noteworthy that the subunits of arsenite oxidases in A. pernix and S. tokodaii lack the cluster stabilizing disulfide bond (fig. 1b), which, according to standard nomenclature (Schmidt and Shaw 2001), would qualify these subunits as Rieske-type rather than genuine Rieske proteins. The Rieske-type proteins so far were only found in bacterial dioxygenases. The fact that the presence/absence of the disulfide bond is obviously not related to the classification into different enzymes, to our mind, renders the distinction between Rieske and Rieske-type proteins obsolete and the term Rieske-type should therefore be avoided in future work. The prominent structural feature of the "proline-loop," well-studied in Rieske/cytb complexes (Carrell et al. 1997), is structurally conserved in the arsenite oxidase Rieske protein from A. faecalis (Ellis et al. 2001). Ironically, in the majority of Rieske proteins from arsenite oxidases, not a single proline is present in the respective sequence stretch and, again, it may be necessary for the sake of clarity to coin a new term for this conserved structural element of Rieske proteins.
The presently available sequences suggest that the first cluster-binding box in the Rieske proteins might serve as a marker sequence distinguishing between representatives of the arsenite oxidase and the Rieske/cytb complex families. In all arsenite oxidases, this sequence reads CXHMG, whereas the methionine residue never occurs in Rieske/cytb complexes.
Most (All?) Arsenite Oxidases Are Membrane-Attached Periplasmic Enzymes
The Rieske subunits of all arsenite oxidases detected in the genomes as well as of those from the two ß-proteobacteria (Mukhopadhyay et al. 2002; Muller et al. 2003) contain an N-terminal hydrophobic stretch similar to the transmembrane helix of the respective subunit in Rieske/cytb complexes. The tat-recognition motif preceeding this hydrophobic stretch is also conserved between the Rieske subunits of the two different enzymes (see fig. 1). Similar to its counterparts in Rieske/cytb complexes (Hinsley et al. 2001), the Rieske subunit of arsenite oxidase must therefore be translocated via the tat system (as also proposed by Muller et al. 2003 and Mukhopadhyay et al. 2002). The catalytic subunit is possibly cotranslated in this step, as is for example the case for the [NiFe] hydrogenases' small and large subunits (Vignais, Billoud, and Meyer 2001).
The presence of an N-terminal hydrophobic stretch in all arsenite oxidases for which gene sequences are available raises the question of whether this stretch is cleaved after the protein has been transported over the cytoplasmic membrane or whether the enzyme remains membrane attached in these species. Several arguments strongly favor the second model. (1) Sulfolobales do not contain a confined periplasmic compartment. Consequently, no truly soluble extracytoplasmic proteins are possible in Sulfolobales. (2) As outlined above, all experimental data suggest a membrane attachment of arsenite oxidase in Chloroflexus. (3) In the ß-proteobacterium C. arsenoxidans, arsenite oxidase activity was found to be associated with membranes (Muller et al. 2003).
Membrane attachment as a general feature of arsenite oxidases is seemingly in conflict with the fact that the enzyme from A. faecalis has been isolated as a soluble complex. However, purification of the Alcaligenes enzyme involves a heating step to 60°C (Anderson, Williams, and Hille 1992), which may well have cleaved the soluble domain of the Rieske protein off its membrane anchor. A proteolytic lability of the hinge region between the transmembrane helix and the soluble domain has previously been observed for the homologous protein in the Rieske/cytb complexes (Carrell et al. 1997). The ensemble of these data therefore strongly suggests that arsenite oxidases are membrane-attached enzymes in all species considered in this work.
A Structural Model for the Membrane Attachment
The Rieske center's g-tensor directions determined in Chloroflexus (fig. 3a), together with the attribution of paramagnetic to molecular axes in Rieske proteins (Schoepp et al. 1999; Brugna et al. 2000) and the presence of a three-dimensional structure for arsenite oxidase (Ellis et al. 2001) allows a structural model for the enzyme's membrane association to be proposed. As shown in figure 3a, the Rieske center's gx direction lies at an angle of 45° with respect to the membrane, whereas its gy axis is parallel to the membrane. The gx direction of Rieske centers has been demonstrated to be collinear with the Fe-Fe direction of the [2Fe-2S] cluster (Brugna et al. 2000), whereas the attribution of gy to either the vector connecting the acid labile sulfur atoms or to the remaining orthogonal direction is still not definitively settled. Taking this gz/gy uncertainty into account, four orientations of the enzyme with respect to the membrane plane are possible. The two that point the exposed (histidine ligated) Fe atom of the Rieske cluster towards the membrane must be discarded since they would place the large subunit within the membrane. Figure 3b and c show the remaining two possible geometries of the whole enzyme with respect to the membrane. Remarkably, both conformations place the N-terminally oriented end of the Rieske protein's chain, which has to connect up to the linker region and ultimately to the transmembrane helix, in proximity of the membrane. A definitive correlation of the g-tensor's gz and gy directions to molecular axes will ultimately allow the removal of the residual ambiguity shown in figure 3b and c.
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Arsenite Oxidase Is a "Pre-LUCA" Enzyme
Both subunits of arsenite oxidase have homologs in functionally unrelated enzymes. The respective family relationships are mutually exclusive, that is, no other enzymes with Rieske subunits also containing molybdopterin subunits and vice versa are known so far. Thus, arsenite oxidase is an exemplary case of an enzyme put together by picking out required redox domains from a restricted set of redox protein building blocks as described recently (Baymann et al. 2003). The respective homologues of arsenite oxidase's Rieske and molybdopterin subunits can be exploited for rooting the phylogenetic tree of arsenite oxidase as depicted in figure 2a and b. Both the phylograms of the Rieske proteins and the phylograms of the molybdopterin subunits show a clear-cut diversification between arsenite oxidases on one side and the remaining enzymes on the other side (i.e., the arsenite oxidase subunits form distinct subtrees in these phylograms). The roots of these subtrees lie in between the archaeal and the bacterial representatives (fig. 2a and b), and the phylogeny of arsenite oxidase therefore corresponds to that of the parent species. This similarity of topology extends to the details of the bacterial part of arsenite oxidase's phylogenetic trees featuring Chloroflexus as an early branching entry in line with 16S rRNA trees (Olsen, Woese, and Overbeek 1994). As discussed in more detail in Baymann et al. 2003, this indicates that an arsenite oxidasetype enzyme was present before the divergence of Bacteria and Archaea. The evolutionary history of arsenite oxidase therefore seems to date back to the era before the existence of the last universal common ancestor (LUCA), making arsenite oxidase part of the electron transfer enzymes that appear to have been assembled from the basic redox building blocks during the very early stages of the evolution of life on earth. The finding of a pre-LUCA origin of these mostly bioenergetic enzymes is particularily intriguing in the light of a novel hypothesis on the origin of life recently put forward by Russell and Hall (1997) and Russell, Hall, and Mellersh (2003) and extended by Martin and Russell (2003). According to this scenario, the earliest "cellular" structures may have been cavities in colloidal FeS precipitates of ocean floor hydrothermal systems. A good deal of the basic metabolic processes (see Martin and Russell 2003) would have evolved in these structures, and it was not before the replacement of the inorganic walls by lipids that free-living cells came into being. According to Martin and Russell, two independent lineages would have invented fundamentally different lipids and biosynthetic pathways thereof, eventually giving birth to the archaeal and the bacterial domains of prokaryotes. One of the main elements of this model, that is, the development of full-fledged metabolic systems already within the mineral-confined, quasicellular structures, therefore predicts that many bioenergetic enzyme systems should have existed in this early phase before the Archaea/Bacteria diversification. Electron transfer utilizing arsenite as electron donor might well be among these "ancient" bioenergetic pathways.
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Acknowledgements |
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Footnotes |
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William Martin, Associate Editor
E-mail: nitschke{at}ibsm.cnrs-mrs.fr.
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Literature Cited |
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Albouy, D., P. Joliot, B. Robert, and W. Nitschke. 1997. Electron transfer towards the RCI-type photosystem in the green sulphur bacterium Chlorobium limicola forma thiosulfatophilum studied by time-resolved optical spectroscopy in-vivo. Eur. J. Biochem. 249:630-636.[Abstract]
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][ISI][Medline]
Anderson, G. L., J. Williams, and R. Hille. 1992. The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. J. Biol.Chem. 267:23674-23682.
Baymann, F., E. Lebrun, M. Brugna, B. Schoepp, M.-T. Giudici-Orticoni, and W. Nitschke. 2003. The redox protein construction kit; pre-LUCA evolution of energy conserving enzymes. Phil. Trans. R. Soc. Lond. B 358:267-274.
Brugna, M. 1997. Evolution du complexe bc: Etude de cette enzyme chez la bactérie primitive Chloroflexus aurantiacus. Diplome d'Etudes Approfondies, Université d'Aix-Marseille I, France.
Brugna, M., D. Albouy, and W. Nitschke. 1998. Diversity of cytochrome bc-complexes: example of the Rieske protein in green sulfur bacteria. J. Bacteriol. 180:3719-3723.
Brugna, M., W. Nitschke, M. Asso, B. Guigliarelli, D. Lemesle-Meunier, and C. Schmidt. 1999. Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. II. The Rieske protein of phylogenetically distant acidophilic organisms. J. Biol. Chem. 274:16766-16772.
Brugna, M., S. Rodgers, A. Schricker, G. Montoya, M. Kazmeier, W. Nitschke, and I. Sinning. 2000. A spectroscopic method for observing the domain movement of the Rieske iron-sulfur protein. Proc. Natl. Acad. Sci. USA 97:2069-2074.
Carrell, C. J., H. Zhang, W. A. Cramer, and J. L. Smith. 1997. Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein. Structure 5:1613-1625.[ISI][Medline]
Ellis, P. J., T. Conrads, R. Hille, and P. Kuhn. 2001. Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 Å and 2.03 Å. Structure 9:125-132.[ISI][Medline]
Hinsley, A. P., N. R. Stanley, T. Palmer, and B. C. Berks. 2001. A naturally occurring bacterial Tat signal peptide lacking one of the invariant arginine residues of the consensus targeting motif. FEBS Lett. 497:45-49.[CrossRef][ISI][Medline]
Jackson, C. R., H. W. Langner, J. Doahoe-Christiansen, W. P. Inskeep, and T. R. McDermott. 2001. Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring. Environ. Microbiol. 3:532-542.[CrossRef][ISI][Medline]
Jormakka, M., S. Toernroth, B. Byrne, and S. Iwata. 2002. Molecular basis of proton motive force generation: structure of formate dehydrogenase-N. Science 295:1863-1868.
Kramer, D. M., B. Schoepp, U. Liebl, and W. Nitschke. 1997. Cyclic electron transfer in Heliobacillus mobilis involving a menaquinol oxidizing cytochrome bc complex and an RCI-type reaction center. Biochemistry 36:4203-4211.[CrossRef][ISI][Medline]
Martin, W., and M. J. Russell. 2003. On the origin of cells: a hypothesis for the evolutionary transitions from prebiotic geochemistry to membrane-bounded chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells. Phil. Trans. R. Soc. Lond. B 358:59-85.
Mukhopadhyay, R., B. P. Rosen, L. T. Phung, and S. Silver. 2002. Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol. Rev. 26:311-325.[CrossRef][ISI][Medline]
Muller, D., D. Lièvremont, D. D. Simeonova, J.-C. Hubert, and M.-C. Lett. 2003. Arsenite oxidase aox genes from a metal-resistant ß-proteobacterium. J. Bact 185:135-141.
Olsen, G. J., C. R. Woese, and R. Overbeek. 1994. The winds of (evolutionary) change: breathing new life into microbiology. J. Bacteriol. 176:1-6.[ISI][Medline]
Russell, M. J., and A. J. Hall. 1997. The emergence of life from iron monosulphide bubbles at a submarine hydrothermal redox and pH front. J. Geol. Soc. Lond. 154:377-402.[ISI][Medline]
Russell, M. J., A. J. Hall, and A. R. Mellersh. 2003. On the dissipation of thermal and chemical energies on the early Earth: the onsets of hydrothermal convection, chemiosmosis, genetically regulated metabolism and oxygenic photosynthesis. Natural and laboratory simulated thermal chemical processes. (in press).
Rutherford, A. W., and P. Sétif. 1990. Orientation of P700, the primary electron donor of photosystem I. Biochim. Biophys. Acta 1100:128-132.
Schmidt, C., and L. Shaw. 2001. A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulphur proteins. J. Bioenerg. Biomembr. 33:9-26.[CrossRef][ISI][Medline]
Schoepp, B., M. Brugna, A. Riedel, W. Nitschke, and D. M. Kramer. 1999. The QO-site inhibitor DBMIB favours the proximal position of the chloroplast Rieske protein and induces a pK-shift of the redox-linked proton. FEBS Lett. 450:245-251.[CrossRef][ISI][Medline]
Schütz, M., M. Brugna, and E. Lebrun, et al. (9 co-authors). 2000. Early evolution of cytochrome bc complexes. J. Mol. Biol. 300:663-675.[CrossRef][ISI][Medline]
Thomson, J. D., T. J. Gilson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 24:4876-4882.[CrossRef]
van Vliet, P. Z. D., W. Nitschke, and A. W. Rutherford. 1991. Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR. Eur. J. Biochem. 199:317-323.[Abstract]
Vignais, P. M., B. Billoud, and J. Meyer. 2001. Classification and phylogeny of hydrogenases. FEMS Microbiol. Rev. 25:455-501.[CrossRef][ISI][Medline]
Wynn, R. M., T. E. Redlinger, J. M. Foster, R. E. Blankenship, R. C. Fuller, R. W. Shaw, and D. B. Knaff. 1987. Electron-transport chains of phototrophically and chemotrophically grown Chloroflexus aurantiacus. Biochim. Biophys. Acta 891:216-226.[ISI]
Yanyushin, M. F. 2002. Fractionation of cytochromes of phototrophically grown Chloroflexus aurantiacus. Is there a cytochrome bc complex among them? FEBS Lett. 512:125-128.[CrossRef][ISI][Medline]
Zannoni, D., and W. J. Ingledew. 1985. A thermodynamic analysis of the plasma membrane electron transport components in photoheterotrophically grown cells of Chloroflexus aurantiacus: an optical and electron paramagnetic resonance study. FEBS Lett. 193:93-98.[CrossRef][ISI]