The Elusive Third Subunit IIa of the Bacterial B-Type Oxidases: The Enzyme from the Hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba3-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix “subunit IIa”, which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA2) of 59000 Da, subunit II (encoded by coxB2) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes

Microbe Profile: Aquifex aeolicus: an extreme heat-loving bacterium that feeds on gases and inorganic chemicals

International audienceHabitat and fundamental metabolic processes of 'Aquifex aeolicus'. Natural living environment: Aquificae are ubiquitous and profuse in both marine and terrestrial hydrothermal systems, including underwater volcanoes and hot springs. They are predominant in many microbial communities in thermal habitats containing filamentous biomass and in sediments of springs, but are also found in planktonic populations. As chemolithoautotrophs, they play a principal role in the biogeochemical cycles of carbon, sulphur, and nitrogen. 'Aquifex aeolicus' is a marine bacterium isolated near the shallow submarine volcanic vents of the island Vulcano (Italy). It feeds on molecular hydrogen, carbon dioxide, molecular oxygen, and mineral nutrients, releasing only water. Instead of H 2 , inorganic sulphur compounds can also be oxidized, up to sulphate. Energy conservation and carbon assimilation: The respiratory chains in 'A. aeolicus' include hydrogenases (Hase I and Hase II), O 2 reductases (ba 3 and bd oxidases), a number of sulphur oxidoreductase enzymes, and the lipid-soluble quinone 2-demethylmenaquinone-7 (DMK 7). The physiological role of complex I, which might function in reverse to produce NADH, remains to be clarified [1]. An electrochemical ion gradient is used for ATP synthesis, motility or other cellular functions. In 'A. aeolicus', the polar flagellar motor is driven by Na + ions. Biomass components are produced from inorganic carbon (the CO 2 fixing pathways are boxed in grey) via an ancestral form of the reductive tricarboxylic acid cycle (rTCA) and a still-uncharacterized reductive folate pathway (incomplete Wood-Ljungdahl pathway) [2]. The rTCA cycle is driven by a pool of low-potential, reduced ferredoxins (Fd red), the reduction of which is still enigmatic in 'A. aeolicus'

Hydrogen metabolism in the hyperthermophilic bacterium Aquifex aeolicus

International audienceAquifex aeolicus is a microaerophilic, hydrogen-oxidizing, hyperthermophilic bacterium containing three [NiFe] hydrogenases. Two of these three enzymes (one membrane-bound and one soluble) have been purified and characterized. The Aquifex hydrogenases are thermostable and tolerant to oxygen. A cellular function for the three hydrogenases has been proposed. The two membrane-bound periplasmic hydrogenases may function in energy conservation, whereas the soluble cytoplasmic hydrogenase is probably involved in the CO2 fixation pathway

Electrophoretic mobility of a monotopic membrane protein inserted into the top of supported lipid bilayers

International audienceWe have studied the translational migration of a monotopic membrane protein, the bacterial sulfide quinone reductase (SQR) in supported n-bilayers (1≤n≲500) under the influence of an electric field parallel to the membrane plane. The direction of the migration changes when the charge of the protein changes its sign. Measuring mobilities at different pH enables us to gain experimental physico-chemical data on SQR as its isoelectric point and its estimated oligomeric state (at least trimeric) when inserted in a lipid membrane. Consequently, in addition to the migration study of membrane proteins in a lipid environment, this experimental system, previously used with a transmembrane protein, is thus suitable to define membrane protein properties in conditions approaching the native ones (in the absence of detergent)

Electrochemistry, AFM and PM-IRRAS spectroscopy of immobilized hydrogenase: role of a trans-membrane helix on enzyme orientation for efficient H2 oxidation

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First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus.

International audienceSulfur oxygenase reductase (SOR) enzyme is responsible for the initial oxidation step of elemental sulfur in archaea. Curiously, Aquifex aeolicus, a hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium, has the SOR-encoding gene in its genome. We showed, for the first time the presence of the SOR enzyme in A. aeolicus, its gene was cloned and recombinantly expressed in Escherichia coli and the protein was purified and characterised. It is a 16 homo-oligomer of approximately 600 kDa that contains iron atoms indispensable for the enzyme activity. The optimal temperature of SOR activity is 80 degrees C and it is inactive at 20 degrees C. Studies of the factors involved in getting the fully active molecule at high temperature show clearly that (1) incubation at high temperature induces more homogeneous form of the enzyme, (2) conformational changes observed at high temperature are required to get the fully active molecule and (3) acquisition of an active conformation induced by the temperature seems to be more important than the subunit number. Differences between A. aeolicus SOR and the archaea SORs are described

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers

International audienceMonotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological syste