Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.     

Heterologously expressed arsenite oxidase: A system to study biogenesis and structure/function relationships of the enzyme family

Studies of native arsenite oxidases from Ralstonia sp. S22 and Rhizobium sp. NT-26 raised two major questions. The first one concerns the mode of the enzyme's membrane-association. It has been suggested that a hypothetical not conserved protein could account for this variable association. Expression of the wild type arsenite oxidase in Escherichia coli allowed us to study the cellular localization of this enzyme in the absence of such a hypothetical partner. The results with the Ralstonia sp. S22 enzyme suggest that no additional protein is required for membrane association. The second question addresses the influence of the disulfide bridge in the small Rieske subunit, conspicuously absent in the Rhizobium sp. NT-26 enzyme, on the properties of the [2Fe-2S] center. The disulfide bridge is considered to be formed only after translocation of the enzyme to the periplasm. To address this question we thus first expressed the enzyme in the absence of its Twin-arginine translocation signal sequence. The spectral and redox properties of the cytoplasmic enzyme are unchanged compared to the periplasmic one. We finally studied a disulfide bridge mutant, Cys106Ala, devoid of the first Cys involved in the disulfide bridge formation. This mutation, proposed to have a strong effect on redox and catalytic properties of the Rieske protein in Rieske/cytb complexes, had no significant effect on properties of the Rieske protein from arsenite oxidase. Our present results demonstrate that the effects attributed to the disulfide bridge in the Rieske/cytb complexes are likely to be secondary effects due to conformational changes.     

Enzyme phylogenies as markers for the oxidation state of the environment: The case of respiratory arsenate reductase and related enzymes

Background  

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far.     

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc ₁ complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc ₁ complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.     

Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A

BACKGROUND: Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation. RESULTS: Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site. CONCLUSIONS: The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

The Small Subunit AroB of Arsenite Oxidase: LESSONS ON THE [2Fe-2S] RIESKE PROTEIN SUPERFAMILY

Here, we describe the characterization of the [2Fe-2S] clusters of arsenite oxidases from Rhizobium sp. NT-26 and Ralstonia sp. 22. Both reduced Rieske proteins feature EPR signals similar to their homologs from Rieske-cyt b complexes, with g values at 2.027, 1.88, and 1.77. Redox titrations in a range of pH values showed that both [2Fe-2S] centers have constant E_m values up to pH 8 at ∼+210 mV. Above this pH value, the E_m values of both centers are pH-dependent, similar to what is observed for the Rieske-cyt b complexes. The redox properties of these two proteins, together with the low E_m value (+160 mV) of the Alcaligenes faecalis arsenite oxidase Rieske (confirmed herein), are in line with the structural determinants observed in the primary sequences, which have previously been deduced from the study of Rieske-cyt b complexes. Since the published E_m value of the Chloroflexus aurantiacus Rieske (+100 mV) is in conflict with this sequence analysis, we re-analyzed membrane samples of this organism and obtain a new value (+200 mV). Arsenite oxidase activity was affected by quinols and quinol analogs, which is similar to what is found with the Rieske-cyt b complexes. Together, these results show that the Rieske protein of arsenite oxidase shares numerous properties with its counterpart in the Rieske-cyt b complex. However, two cysteine residues, strictly conserved in the Rieske-cyt b-Rieske and considered to be crucial for its function, are not conserved in the arsenite oxidase counterpart. We discuss the role of these residues.     

Look on the positive side! The orientation, identification and bioenergetics of 'Archaeal' membrane-bound nitrate reductases

Many species of Bacteria and Archaea respire nitrate using a molybdenum-dependent membrane-bound respiratory system called Nar. Classically, the 'Bacterial' Nar system is oriented such that nitrate reduction takes place on the inside of this membrane. However, the active site subunit of the 'Archaeal' Nar systems has a twin arginine ('RR') motif, which is a suggestion of translocation to the outside of the cytoplasmic membrane. These 'Archaeal' type of nitrate reductases are part of a group of molybdoenzymes with an 'RR' motif that are predicted to have an aspartate ligand to the molybdenum ion. This group includes selenate reductases and possible sequence signatures are described that serve to distinguish the Nar nitrate reductases from the selenate reductases. The 'RR' sequences of nitrate reductases of Archaea and some that have recently emerged in Bacteria are also considered and it is concluded that there is good evidence for there being both Archaeal and Bacterial examples of Nar-type nitrate reductases with an active site on the outside of the cytoplasmic membrane. Finally, the bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic membrane have been explored.     

The proportional lack of archaeal pathogens: Do viruses/phages hold the key?

Although Archaea inhabit the human body and possess some characteristics of pathogens, there is a notable lack of pathogenic archaeal species identified to date. We hypothesize that the scarcity of disease-causing Archaea is due, in part, to mutually-exclusive phage and virus populations infecting Bacteria and Archaea, coupled with an association of bacterial virulence factors with phages or mobile elements. The ability of bacterial phages to infect Bacteria and then use them as a vehicle to infect eukaryotes may be difficult for archaeal viruses to evolve independently. Differences in extracellular structures between Bacteria and Archaea would make adsorption of bacterial phage particles onto Archaea (i.e. horizontal transfer of virulence) exceedingly hard. If phage and virus populations are indeed exclusive to their respective host Domains, this has important implications for both the evolution of pathogens and approaches to infectious disease control.     

The enigma of ribonuclease P evolution

The 5'-end maturation of tRNAs is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P) in all organisms. Here we provide, for the first time, a comprehensive overview on the representation of individual RNase P protein homologs within the Eukarya and Archaea. Most eukaryotes have homologs for all four protein subunits (Pop4, Rpp1, Pop5 and Rpr2) present in the majority of Archaea. Pop4 is the only RNase P protein subunit identifiable in all Eukarya and Archaea with available genome sequences. Remarkably, there is no structural homology between bacterial and archaeal-eukaryotic RNase P proteins. The simplest interpretation is that RNase P has an 'RNA-alone' origin and progenitors of Bacteria and Archaea diverged very early in evolution and then pursued completely different strategies in the recruitment of protein subunits during the transition from the 'RNA-alone' to the 'RNA-protein' state of the enzyme.     

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.     

Eukaryotic ribonuclease P: increased complexity to cope with the nuclear pre-tRNA pathway

Ribonuclease P is an ancient enzyme that cleaves pre-tRNAs to generate mature 5' ends. It contains an essential RNA subunit in Bacteria, Archaea, and Eukarya, but the degree to which the RNA subunit relies on proteins to supplement catalysis is highly variable. The eukaryotic nuclear holoenzyme has recently been found to contain almost twenty times the protein content of the bacterial enzymes, in addition to having split into at least two related enzymes with distinct substrate specificity. In this review, recent progress in understanding the molecular architecture and functions of nuclear forms of RNase P will be considered.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

About the last common ancestor, the universal life-tree and lateral gene transfer: a reappraisal

An organismal tree rooted in the bacterial branch and derived from a hyperthermophilic last common ancestor (LCA) is still widely assumed to represent the path followed by evolution from the most primeval cells to the three domains recognized among contemporary organisms: Bacteria, Archaea and Eucarya. In the past few years, however, more and more discrepancies between this pattern and individual protein trees have been brought to light. There has been an overall tendency to attribute these incongruities to widespread lateral gene transfer. However, recent developments, a reappraisal of earlier evidence and considerations of our own lead us to a quite different view. It would appear (i) that the role of lateral gene transfer was overemphasized in recent discussions of molecular phylogenies; (ii) that the LCA was probably a non-thermophilic protoeukaryote from which both Archaea and Bacteria emerged by reductive evolution but not as sister groups, in keeping with a current evolutionary scheme for the biosynthesis of membrane lipids; and (iii) that thermophilic Archaea may have been the first branch to diverge from the ancestral line.     

Evidence that the hydrophobic domain of rat renal γ-glutamyltransferase spans the brush border membrane

Lactoperoxidase and glucose oxidase catalyzed ¹²⁵I-iodination was used to specifically label isolated rat renal brush border membrane vesicles from either side of the membrane. Autoradiography of total membrane proteins demonstrated that asymmetric labeling was achieved. Specific immunoprecipitates of aminopeptidase M, an established transmembrane protein, and of γ-glutamyltransferase were isolated from vesicles solubilized with Triton X-100 or with papain. Following electrophoresis and autoradiography, the immunoprecipitates of the two solubilized forms of each enzyme derived from externally labeled vesicles exhibited the same intensity of labeling. In these experiments, the small subunit of the γ-glutamyltransferase was preferentially labeled suggesting that, compared to the large subunit, it is more exposed on the external surface of the membrane. With the samples derived from internally labeled vesicles, the Triton-solubilized form of each enzyme was intensely labeled, whereas the papain-solubilized forms contained insignificant amounts of radioactivity. Thus, the extent of contramembrane labeling was minimal. In these experiments, the large subunit of the γ-glutamyltransferase was preferentially labeled. The similarity of the labeling patterns obtained for aminopeptidase M and γ-glutamyltransferase suggests that the hydrophobic domain of the two amphipathic enzymes are selectively labeled from the internal surface and that the γ-glutamyltransferase may also be a transmembrane protein.     

Zelloberflächenstrukturen der Bacteria und Archaea

Cell wall types of Bacteria and Archaea The acaryote microorganisms are divided into the two domains Bacteria and Archaea. The third domain represent the Eukarya. There is no universal cell wall polymer found in all Bacteria and Archaea. Due to their morphology several cell wall types can be identified, but the chemical diversity of the individual polymers is considerably greater. Certain cell wall polymers are limited to one of the two domains of Bacteria or Archaea like the murein of the Bacteria or the pseudomurein of some methanogens. Peptidoglycans (murein, pseudomurein) do not occur in eukaryotes. On the other hand individual cell wall polymers possess similarities to polymers of other domains. The structural principle of the methanochondroitin is also implemented in the eukaryotic connective tissue. The cell wall polymers consist frequently of glycoconjugates in which the amino acid content (glycoproteins) or the glycan moiety (proteoglycan‐like polymers) predominate. Both components (carbohydrates, amino acids) can also occur in similar amounts (peptidoglycan). There exist also cell wall polymers, which consist only of glycans (slimes, methanochondroitin) or amino acids (proteins, poly‐γ‐D‐glutamyl polymers). Cell wall‐free species (Mycoplasma) also occur. The chemical composition of the cell surface polymers was one of the first phenotypic characteristics that supported the 16 sRNA concept of Carl Woese to assign acaryote organisms into the two domains Bacteria and Archaea. A common feature of all Archaea is the lack of muramic acid and an outer membrane. The later occurs in the gramnegative Bacteria. During the evolution of Bacteria and Archaea a great variety of chemically different cell wall polymers has been developed which allow the growth and interaction of Bacteria and Archaea in different habitats. In this paper, some important surface polymers of Bacteria and Archaea are presented according to their chemical composition.     

From pre-cells to Eukarya--a tale of two lipids

A mechanistic hypothesis for the origin of the three domains of life is proposed. A population of evolving pre-cells is suggested to have had a membrane of a racemate of chiral lipids that continuously underwent spontaneous symmetry breaking by spatial phase segregation into two enantiomerically enriched membrane domains. By frequent pre-cell fusions and fissions these membrane domains became partitioned between two pre-cell subpopulations having predominantly one lipid enantiomer or the other. The origin of the Bacteria and Archaea is explained by divergence of first a population of proto-bacteria and later a population of proto-archaea from the evolving pre-cells, each by the emergence of an enantio-selective lipid biosynthesis within the corresponding pre-cell subtype. The origin of the Eukarya is explained by symbiosis between a population of Bacteria and a subpopulation of pre-cells with a predominance of the bacteria-type lipid enantiomers.