Bacterial Respiratory Chain Diversity Reveals a Cytochrome c Oxidase Reducing O2 at Low Overpotentials.

Cytochrome c oxidases (CcOs) are the terminal enzymes in energy-converting chains of microorganisms, where they reduce oxygen into water. Their affinity for O2 makes them attractive biocatalysts for technological devices in which O2 concentration is limited, but the high overpotentials they display on electrodes severely limit their applicative use. Here, the CcO of the acidophilic bacterium Acidithiobacillus ferrooxidans is studied on various carbon materials by direct protein electrochemistry and mediated one with redox mediators either diffusing or co-immobilized at the electrode surface. The entrapment of the CcO in a network of hydrophobic carbon nanofibers permits a direct electrochemical communication between the enzyme and the electrode. We demonstrate that the CcO displays a µM affinity for O2 and reduces O2 at exceptionally high electrode potentials in the range of +700 to +540 mV vs NHE over a pH range of 4-6. The kinetics of interactions between the enzyme and its physiological partners are fully quantified. Based on these results, an electron transfer pathway allowing O2 reduction in the acidic metabolic chain is proposed.

Impact of copper ligand mutations on a cupredoxin with a green copper center.

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

Scaffoldin modules serving as "cargo" domains to promote the secretion of heterologous cellulosomal cellulases by Clostridium acetobutylicum.

The secretion of large heterologous cellulases by Clostridium acetobutylicum was formerly shown to be deleterious. To circumvent this issue, various scaffoldins' modules were grafted at their N termini. Family 3a cellulose binding module combined with an X2 module(s) was found to trigger the secretion of Clostridium cellulolyticum cellulases by the solventogenic bacterium.

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.

Fermentative hydrogen production in an up-flow anaerobic biofilm reactor inoculated with a co-culture of Clostridium acetobutylicum and Desulfovibrio vulgaris.

Dark fermentation systems often show low H2 yields and unstable H2 production, as the result of the variability of microbial dynamics and metabolic pathways. Recent batch investigations have demonstrated that an artificial consortium of two anaerobic bacteria, Clostridium acetobutylicum and Desulfovibrio vulgaris Hildenborough, may redirect metabolic fluxes and improve H2 yields. This study aimed at evaluating the scale-up from batch to continuous H2 production in an up-flow anaerobic packed-bed reactor (APBR) continuously fed with a glucose-medium. The effects of various parameters, including void hydraulic retention time (HRTv), pH, and alkalinity, on H2 production performances and metabolic pathways were investigated. The results demonstrated that a stable H2 production was reached after 3-4days of operation. H2 production rates increased significantly with decreasing HRTv from 4 to 2h. Instead, H2 yields remained almost stable despite the change in HRTv, indicating that the decrease in HRTv did not affect the global metabolism.

Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase.

Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed.

Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus.

The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus, a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (∼240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite.

A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

Spectroscopic characterization of a green copper site in a single-domain cupredoxin.

Cupredoxins are widespread copper-binding proteins, mainly involved in electron transfer pathways. They display a typical rigid greek key motif consisting of an eight stranded ß-sandwich. A fascinating feature of cupredoxins is the natural diversity of their copper center geometry. These geometry variations give rise to drastic changes in their color, such as blue, green, red or purple. Based on several spectroscopic and structural analyses, a connection between the geometry of their copper-binding site and their color has been proposed. However, little is known about the relationship between such diversity of copper center geometry in cupredoxins and possible implications for function. This has been difficult to assess, as only a few naturally occurring green and red copper sites have been described so far. We report herein the spectrocopic characterization of a novel kind of single domain cupredoxin of green color, involved in a respiratory pathway of the acidophilic organism Acidithiobacillus ferrooxidans. Biochemical and spectroscopic characterization coupled to bioinformatics analysis reveal the existence of some unusual features for this novel member of the green cupredoxin sub-family. This protein has the highest redox potential reported to date for a green-type cupredoxin. It has a constrained green copper site insensitive to pH or temperature variations. It is a green-type cupredoxin found for the first time in a respiratory pathway. These unique properties might be explained by a region of unknown function never found in other cupredoxins, and by an unusual length of the loop between the second and the fourth copper ligands. These discoveries will impact our knowledge on non-engineered green copper sites, whose involvement in respiratory chains seems more widespread than initially thought.

Study of the high-potential iron sulfur protein in Halorhodospira halophila confirms that it is distinct from cytochrome c as electron carrier.

The role of high-potential iron sulfur protein (HiPIP) in donating electrons to the photosynthetic reaction center in the halophilic gamma-proteobacterium Halorhodospira halophila was studied by EPR and time-resolved optical spectroscopy. A tight complex between HiPIP and the reaction center was observed. The EPR spectrum of HiPIP in this complex was drastically different from that of the purified protein and provides an analytical tool for the detection and characterization of the complexed form in samples ranging from whole cells to partially purified protein. The bound HiPIP was identified as iso-HiPIP II. Its Em value at pH 7 in the form bound to the reaction center was approximately 100 mV higher (+140 +/- 20 mV) than that of the purified protein. EPR on oriented samples showed HiPIP II to be bound in a well defined geometry, indicating the presence of specific protein-protein interactions at the docking site. At moderately reducing conditions, the bound HiPIP II donates electrons to the cytochrome subunit bound to the reaction center with a half-time of < or =11 micros. This donation reaction was analyzed by using Marcus's outer-sphere electron-transfer theory and compared with those observed in other HiPIP-containing purple bacteria. The results indicate substantial differences between the HiPIP- and the cytochrome c2-mediated re-reduction of the reaction center.