New insights into the respiratory chains of the chemolithoautotrophic and hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus, a highly hyperthermophilic bacterium, grows chemolithoautotrophically at 85 degrees C, with hydrogen as electron donor and oxygen as electron acceptor in the presence of a sulfur compound. Stimulated by its exceptional physiological properties, we have set out to study the oxygen metabolism of this microorganism. With the use of an unconventional integrative proteomic approach combining separation of membrane proteins by Blue-Native electrophoresis, detection of enzyme activities in-gel and direct protein identification by two-dimensional liquid chromatography and tandem mass spectrometry (2D nanoLC-MS/MS), we have obtained evidence for the presence of functional respiratory enzymes in membranes of A. aeolicus cultivated with H2/O2/S0 as well as an organization in stable superstructures of some of these individual complexes. This study has revealed the assembly of the bc complex and a cytochrome coxidase as a supercomplex and possible associations of electron transfer proteins and complexes involved in oxygen reduction such as sulfide quinone reductase, cytochrome c oxidase, bc complex, membrane-bound hydrogenase I and quinol oxidase. Electron transfer measurements on solubilized membranes have demonstrated the existence of uncommon respiratory chains (sulfide/oxygen as well as hydrogen/oxygen) in the cell growth conditions used. Moreover, the subunit composition of some of the complexes has been more precisely described, particularly that of complex I, leading for the first time to evidence of the presence of several isoforms of this complex. We can propose from our results (in-gel identification and functional data) that the bioenergetic pathways (sulfur and oxygen reductions) may be organized in supramolecular structures in A. aeolicus, as we have previously purified and characterized a hydrogen-oxidizing sulfur-reducing supercomplex from this bacterium.