Quantitative proteomics reveals the Sox system's role in sulphur and arsenic metabolism of phototroph Halorhodospira halophila.

The metabolic process of purple sulphur bacteria's anoxygenic photosynthesis has been primarily studied in Allochromatium vinosum, a member of the Chromatiaceae family. However, the metabolic processes of purple sulphur bacteria from the Ectothiorhodospiraceae and Halorhodospiraceae families remain unexplored. We have analysed the proteome of Halorhodospira halophila, a member of the Halorhodospiraceae family, which was cultivated with various sulphur compounds. This analysis allowed us to reconstruct the first comprehensive sulphur-oxidative photosynthetic network for this family. Some members of the Ectothiorhodospiraceae family have been shown to use arsenite as a photosynthetic electron donor. Therefore, we analysed the proteome response of Halorhodospira halophila when grown under arsenite and sulphide conditions. Our analyses using ion chromatography-inductively coupled plasma mass spectrometry showed that thioarsenates are chemically formed under these conditions. However, they are more extensively generated and converted in the presence of bacteria, suggesting a biological process. Our quantitative proteomics revealed that the SoxAXYZB system, typically dedicated to thiosulphate oxidation, is overproduced under these growth conditions. Additionally, two electron carriers, cytochrome c551/c5 and HiPIP III, are also overproduced. Electron paramagnetic resonance spectroscopy suggested that these transporters participate in the reduction of the photosynthetic Reaction Centre. These results support the idea of a chemically and biologically formed thioarsenate being oxidized by the Sox system, with cytochrome c551/c5 and HiPIP III directing electrons towards the Reaction Centre.

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α-Helical Metalloprotein.

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.

Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c.

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s-1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.

The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase.

While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n=2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, Ks, of the Mo(V) semi-reduced intermediate is found to be lower than 10(-3). Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable Mo(V) with KS=4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes. Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n=2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed.

Free energy conversion in the LUCA: Quo vadis?

Living entities are unimaginable without means to harvest free energy from the environment, that is, without bioenergetics. The quest to understand the bioenergetic ways of early life therefore is one of the crucial elements to understand the emergence of life on our planet. Over the last few years, several mutually exclusive scenarios for primordial bioenergetics have been put forward, all of which are based on some sort of empirical observation, a remarkable step forward from the previous, essentially untestable, ab initio models. We here try to present and compare these scenarios while at the same time discuss their respective empirical weaknesses. The goal of this article is to harness crucial new expertise from the entire field by stimulating a larger part of the bioenergetics community to become involved in "origin-of-energy-metabolism" research. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Arsenics as bioenergetic substrates.

Although at low concentrations, arsenic commonly occurs naturally as a local geological constituent. Whereas both arsenate and arsenite are strongly toxic to life, a number of prokaryotes use these compounds as electron acceptors or donors, respectively, for bioenergetic purposes via respiratory arsenate reductase, arsenite oxidase and alternative arsenite oxidase. The recent burst in discovered arsenite oxidizing and arsenate respiring microbes suggests the arsenic bioenergetic metabolisms to be anything but exotic. The first goal of the present review is to bring to light the widespread distribution and diversity of these metabolizing pathways. The second goal is to present an evolutionary analysis of these diverse energetic pathways. Taking into account not only the available data on the arsenic metabolizing enzymes and their phylogenetical relatives but also the palaeogeochemical records, we propose a crucial role of arsenite oxidation via arsenite oxidase in primordial life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

The prokaryotic Mo/W-bisPGD enzymes family: a catalytic workhorse in bioenergetic.

Over the past two decades, prominent importance of molybdenum-containing enzymes in prokaryotes has been put forward by studies originating from different fields. Proteomic or bioinformatic studies underpinned that the list of molybdenum-containing enzymes is far from being complete with to date, more than fifty different enzymes involved in the biogeochemical nitrogen, carbon and sulfur cycles. In particular, the vast majority of prokaryotic molybdenum-containing enzymes belong to the so-called dimethylsulfoxide reductase family. Despite its extraordinary diversity, this family is characterized by the presence of a Mo/W-bis(pyranopterin guanosine dinucleotide) cofactor at the active site. This review highlights what has been learned about the properties of the catalytic site, the modular variation of the structural organization of these enzymes, and their interplay with the isoprenoid quinones. In the last part, this review provides an integrated view of how these enzymes contribute to the bioenergetics of prokaryotes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

On the universal core of bioenergetics.

Living cells are able to harvest energy by coupling exergonic electron transfer between reducing and oxidising substrates to the generation of chemiosmotic potential. Whereas a wide variety of redox substrates is exploited by prokaryotes resulting in very diverse layouts of electron transfer chains, the ensemble of molecular architectures of enzymes and redox cofactors employed to construct these systems is stunningly small and uniform. An overview of prominent types of electron transfer chains and of their characteristic electrochemical parameters is presented. We propose that basic thermodynamic considerations are able to rationalise the global molecular make-up and functioning of these chemiosmotic systems. Arguments from palaeogeochemistry and molecular phylogeny are employed to discuss the evolutionary history leading from putative energy metabolisms in early life to the chemiosmotic diversity of extant organisms. Following the Occam's razor principle, we only considered for this purpose origin of life scenarios which are contiguous with extant life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Was nitric oxide the first deep electron sink?

Evolutionary histories of enzymes involved in chemiosmotic energy conversion indicate that a strongly oxidizing substrate was available to the last universal common ancestor before the divergence of Bacteria and Archaea. According to palaeogeochemical evidence, O(2) was not present beyond trace amounts on the early Earth. Based on recent phylogenetic, enzymatic and geochemical results, we propose that, in the earliest Archaean, nitric oxide (NO) and its derivatives nitrate and nitrite served as strongly oxidizing substrates driving the evolution of a bioenergetic pathway related to modern dissimilatory denitrification. Aerobic respiration emerged later from within this ancestral pathway via adaptation of the enzyme NO reductase to its new substrate, dioxygen.

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.

Menaquinone as pool quinone in a purple bacterium.

Purple bacteria have thus far been considered to operate light-driven cyclic electron transfer chains containing ubiquinone (UQ) as liposoluble electron and proton carrier. We show that in the purple gamma-proteobacterium Halorhodospira halophila, menaquinone-8 (MK-8) is the dominant quinone component and that it operates in the Q(B)-site of the photosynthetic reaction center (RC). The redox potentials of the photooxidized pigment in the RC and of the Rieske center of the bc(1) complex are significantly lower (E(m) = +270 mV and +110 mV, respectively) than those determined in other purple bacteria but resemble those determined for species containing MK as pool quinone. These results demonstrate that the photosynthetic cycle in H. halophila is based on MK and not on UQ. This finding together with the unusual organization of genes coding for the bc(1) complex in H. halophila suggests a specific scenario for the evolutionary transition of bioenergetic chains from the low-potential menaquinones to higher-potential UQ in the proteobacterial phylum, most probably induced by rising levels of dioxygen 2.5 billion years ago. This transition appears to necessarily proceed through bioenergetic ambivalence of the respective organisms, that is, to work both on MK- and on UQ-pools. The establishment of the corresponding low- and high-potential chains was accompanied by duplication and redox optimization of the bc(1) complex or at least of its crucial subunit oxidizing quinols from the pool, the Rieske protein. Evolutionary driving forces rationalizing the empirically observed redox tuning of the chain to the quinone pool are discussed.

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 approximately +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.

Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes.

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a beta-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c(554). Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c(552) and c(554,) are from the parent species. Cytochrome c(552) belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus.

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αßγ)2 of about 390 kDa. The KM for sulfite and kcat values were 34 µM and 567 s-1 respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ10 analogue, the decyl-ubiquinone, as well, with a KM of 2.6 µM and a kcat of 52.9 s-1. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase.

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.

The controversy on the ancestral arsenite oxidizing enzyme; deducing evolutionary histories with phylogeny and thermodynamics.

The three presently known enzymes responsible for arsenic-using bioenergetic processes are arsenite oxidase (Aio), arsenate reductase (Arr) and alternative arsenite oxidase (Arx), all of which are molybdoenzymes from the vast group referred to as the Mo/W-bisPGD enzyme superfamily. Since arsenite is present in substantial amounts in hydrothermal environments, frequently considered as vestiges of primordial biochemistry, arsenite-based bioenergetics has long been predicted to be ancient. Conflicting scenarios, however, have been put forward proposing either Arr/Arx or Aio as operating in the ancestral metabolism. Phylogenetic data argue in favor of Aio whereas biochemical and physiological data led several authors to propose Arx/Arr as the most ancient anaerobic arsenite metabolizing enzymes. Here we combine phylogenetic approaches with physiological and biochemical experiments to demonstrate that the Arx/Arr enzymes could not have been functional in the Archaean geological eon. We propose that Arr reacts with menaquinones to reduce arsenate whereas Arx reacts with ubiquinone to oxidize arsenite, in line with thermodynamic considerations. The distribution of the quinone biosynthesis pathways, however, clearly indicates that the ubiquinone pathway is recent. An updated phylogeny of Arx furthermore reinforces the hypothesis of a recent emergence of this enzyme. We therefore conclude that anaerobic arsenite redox conversion in the Archaean must have been performed in a metabolism involving Aio.

Fougerite: the not so simple progenitor of the first cells.

We here review the extraordinary mineralogical properties of green rusts and their naturally occurring form, fougerite, and discuss the pertinence of these properties within the alkaline hydrothermal vent (AHV) hypothesis for life's emergence. We put forward an extended version of the AHV scenario which enhances the conformity between extant life and its earliest progenitor by extensively making use of fougerite's mechanistic and catalytic particularities.

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. RESULTS: We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. CONCLUSION: These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

On the Natural History of Flavin-Based Electron Bifurcation.

Electron bifurcation is here described as a special case of the continuum of electron transfer reactions accessible to two-electron redox compounds with redox cooperativity. We argue that electron bifurcation is foremost an electrochemical phenomenon based on (a) strongly inverted redox potentials of the individual redox transitions, (b) a high endergonicity of the first redox transition, and (c) an escapement-type mechanism rendering completion of the first electron transfer contingent on occurrence of the second one. This mechanism is proposed to govern both the traditional quinone-based and the newly discovered flavin-based versions of electron bifurcation. Conserved and variable aspects of the spatial arrangement of electron transfer partners in flavoenzymes are assayed by comparing the presently available 3D structures. A wide sample of flavoenzymes is analyzed with respect to conserved structural modules and three major structural groups are identified which serve as basic frames for the evolutionary construction of a plethora of flavin-containing redox enzymes. We argue that flavin-based and other types of electron bifurcation are of primordial importance to free energy conversion, the quintessential foundation of life, and discuss a plausible evolutionary ancestry of the mechanism.

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples.

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.