Exploring substrate interaction in respiratory alternative complex III from Rhodothermus marinus.

Rhodothermus marinus is a thermohalophilic organism that has optimized its microaerobic metabolism at 65 °C. We have been exploring its respiratory chain and observed the existence of a quinone:cytochrome c oxidoreductase complex, named Alternative Complex III, structurally different from the bc1 complex. In the present work, we took profit from nanodiscs and liposomes technology to investigate ACIII activity in membrane-mimicking systems. In addition, we studied the interaction of ACIII with menaquinone, its potential electron acceptors (HiPIP and cytochrome c) and the caa3 oxygen reductase.

Proteomic Time-Course Analysis of the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aurantiacus, during the Transition from Respiration to Phototrophy.

Chloroflexus aurantiacus is a filamentous anoxygenic phototrophic bacterium that grows chemotrophically under oxic conditions and phototrophically under anoxic conditions. Because photosynthesis-related genes are scattered without any gene clusters in the genome, it is still unclear how this bacterium regulates protein expression in response to environmental changes. In this study, we performed a proteomic time-course analysis of how C. aurantiacus expresses proteins to acclimate to environmental changes, namely the transition from chemoheterotrophic respiratory to photoheterotrophic growth mode. Proteomic analysis detected a total of 2520 proteins out of 3934 coding sequences in the C. aurantiacus genome from samples collected at 13 time points. Almost all proteins for reaction centers, light-harvesting chlorosomes, and carbon fixation pathways were successfully detected during the growing phases in which optical densities and relative bacteriochlorophyll c contents increased simultaneously. Combination of proteomics and pigment analysis suggests that the self-aggregation of bacteriochlorophyllide c could precede the esterification of the hydrophobic farnesyl tail in cells. Cytoplasmic subunits of alternative complex III were interchanged between oxic and anoxic conditions, although membrane-bound subunits were used for both conditions. These data highlight the protein expression dynamics of phototrophy-related genes during the transition from respiration to phototrophy.

The Monoheme c Subunit of Respiratory Alternative Complex III Is Not Essential for Electron Transfer to Cytochrome aa3 in Flavobacterium johnsoniae.

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

Ancestry and adaptive radiation of Bacteroidetes as assessed by comparative genomics.

To date, the phylum Bacteroidetes comprises more than 1,500 described species with diverse ecological roles. However, there is little understanding of archetypal Bacteroidetes traits at a genomic level. In this study, a representative set of 89 Bacteroidetes genomes was compiled, and pairwise reciprocal best-match gene comparisons and gene syntenies were used to identify common traits that allowed Bacteroidetes evolution and adaptive radiation to be traced. The type IX secretion system (T9SS) was highly conserved among all studied Bacteroidetes. Class-level comparisons furthermore suggested that the ACIII-caa3COX super-complex evolved in the ancestral aerobic bacteroidetal lineage, and was secondarily lost in extant anaerobic Bacteroidetes. Another Bacteroidetes-specific respiratory chain adaptation was the sodium-pumping Nqr complex I that replaced the ancestral proton-pumping complex I in marine species. T9SS plays a role in gliding motility and the acquisition of complex macro-molecular organic compounds, and the ACIII-caa3COX super-complex allows effective control of electron flux during respiration. This combination likely provided ancestral Bacteroidetes with a decisive competitive advantage to effectively scavenge, uptake and degrade complex organic molecules, and therefore has played a pivotal role in the successful adaptive radiation of the phylum.

Structural basis underlying the electron transfer features of a blue copper protein auracyanin from the photosynthetic bacterium Roseiflexus castenholzii.

Auracyanin (Ac) is a blue copper protein that mediates the electron transfer between Alternative Complex III (ACIII) and downstream electron acceptors in both fort chains of filamentous anoxygenic phototrophs. Here, we extracted and purified the air-oxidized RfxAc from the photoheterotrophically grown Roseiflexus castenholzii, and we illustrated the structural basis underlying its electron transferring features. Spectroscopic and enzymatic analyses demonstrated the reduction of air-oxidized RfxAc by the ACIII upon oxidation of menaquinol-4 and menaquinol-7. Crystal structures of the air-oxidized and Na-dithionite-reduced RfxAc at 2.2 and 2.0 Å resolutions, respectively, showed that the copper ions are coordinated by His77, His146, Cys141, and Met151 in minor different geometries. The Cu1-Sδ bond length increase of Met151, and the electron density Fourier differences at Cu1 and His77 demonstrated their essential roles in the dithionite-induced reduction. Structural comparisons further revealed that the RfxAc contains a Chloroflexus aurantiacus Ac-A-like copper binding pocket and a hydrophobic patch surrounding the exposed edge of His146 imidazole, as well as an Ac-B-like Ser- and Thr-rich polar patch located at a different site on the surface. These spectroscopic and structural features allow RfxAc to mediate electron transfers between the ACIII and redox partners different from those of Ac-A and Ac-B. These results provide a structural basis for further investigating the electron transfer and energy transformation mechanism of bacterial photosynthesis, and the diversity and evolution of electron transport chains.

Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs.

Styrene-maleic acid (SMA) copolymers can extract membrane proteins from native membranes along with lipids as nanodiscs. Preparation with SMA is fast, cost-effective, and captures the native protein-lipid interactions. On the other hand, cryo-EM has become increasingly successful and efficient for structural determinations of membrane proteins, with biochemical sample preparation often the bottleneck. Three recent cryo-EM studies on the efflux transporter AcrB and the alternative complex III: cyt c oxidase supercomplex have demonstrated the potential of SMA nanodisc samples to yield high-resolution structure information of membrane proteins.

Supramolecular organization of photosynthetic complexes in membranes of Roseiflexus castenholzii.

The photosynthetic membranes of the filamentous anoxygenic phototroph Roseiflexus castenholzii have been studied with electron microscopy, atomic force microscopy, and biochemistry. Electron microscopy of the light-harvesting reaction center complex produced a 3D model that aligns with the solved crystal structure of the RC-LH1 from Thermochromatium tepidum with the H subunit removed. Atomic force microscopy of the whole membranes yielded a picture of the supramolecular organization of the major proteins in the photosynthetic electron transport chain. The results point to a loosely packed membrane without accessory antenna proteins or higher order structure.

Alternative Complex III from phototrophic bacteria and its electron acceptor auracyanin.

Alternative Complex III (ACIII) is a multisubunit integral membrane protein electron transfer complex that is proposed to be an energy-conserving functional replacement for the bacterial cytochrome bc1 or b6f complexes. Clues to the structure and function of this novel complex come from its relation to other bacterial enzyme families. The ACIII complex has menaquinone: electron acceptor oxidoreductase activity and contains protein subunits with multiple Fe-S centers and c-type hemes. ACIII is found in a diverse group of bacteria, including both phototrophic and nonphototrophic taxa. In the phototrophic filamentous anoxygenic phototrophs, the electron acceptor is the small blue copper protein auracyanin instead of a soluble cytochrome. Recent work on ACIII and the copper protein auracyanin is reviewed with focus on the photosynthetic systems and potential electron transfer pathways and mechanisms. Taken together, the ACIII complexes constitute a unique system for photosynthetic electron transfer and energy conservation. This article is part of a Special Issue entitled: Respiratory Complex III and related bc complexes.

Structural composition of alternative complex III: variations on the same theme.

Alternative complex III forms a recently identified family of enzymes with quinol:electron acceptor oxidoreductase activity. First biochemical and genomic analyses showed that ACIII is composed of six to eight subunits, most of which homologous to different proteins or domains already observed in other known enzymatic complexes. The increasing number of completely sequenced genomes led us to perform a new search for the genes coding for the different ACIII subunits. We have identified a larger number of gene clusters coding for ACIII, still confined to the bacterial domain, but extended to classes in which it was not observed before. We also found an unanticipated diversity in gene clusters, both in terms of its constitution and organization. The several unexpected gene arrangements brought new perspectives to the role of the different subunits of ACIII, namely in quinone binding and in proton translocation. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.