Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants.

We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I1III2 assembly in bovine heart and the yeast Yarrowia lipolytica, but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III2, but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer.

On the origins of oxygenic photosynthesis and aerobic respiration in Cyanobacteria.

The origin of oxygenic photosynthesis in Cyanobacteria led to the rise of oxygen on Earth ~2.3 billion years ago, profoundly altering the course of evolution by facilitating the development of aerobic respiration and complex multicellular life. Here we report the genomes of 41 uncultured organisms related to the photosynthetic Cyanobacteria (class Oxyphotobacteria), including members of the class Melainabacteria and a new class of Cyanobacteria (class Sericytochromatia) that is basal to the Melainabacteria and Oxyphotobacteria All members of the Melainabacteria and Sericytochromatia lack photosynthetic machinery, indicating that phototrophy was not an ancestral feature of the Cyanobacteria and that Oxyphotobacteria acquired the genes for photosynthesis relatively late in cyanobacterial evolution. We show that all three classes independently acquired aerobic respiratory complexes, supporting the hypothesis that aerobic respiration evolved after oxygenic photosynthesis.

The alternative complex III: a different architecture using known building modules.

Until recently cytochrome bc(1) complexes were the only enzymes known to be able to transfer electrons from reduced quinones to cytochrome c. However, a complex with the same activity and with a unique subunit composition was purified from the membranes of Rhodothermus marinus. This complex, named alternative complex III (ACIII) was then biochemical, spectroscopic and genetically characterized. Later it was observed that the presence of ACIII was not exclusive of R. marinus being the genes coding for ACIII widespread, at least in the Bacteria domain. In this work, a comprehensive description of the current knowledge on ACIII is presented. The relation of ACIII with members of the complex iron-sulfur molybdoenzyme family is investigated by analyzing all the available completely sequenced genomes. It is concluded that ACIII is a new complex composed by a novel combination of modules already identified in other respiratory complexes.

ClassRHO: a platform for classification of bacterial rieske non-heme iron ring-hydroxylating oxygenases.

We have developed an easy-to-use multiplatform classification tool, ClassRHO, which facilitates classification and comparison of bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases (RHOs). Visualization and analysis can be generated on-the-fly by entering or uploading RHO query sequences. Pre-computed classifications were implemented for 42 standard RHO sequences. These 42 RHO sequences can be flexibly selected based on user requests. ClassRHO provides users with many options to view and analyze RHO sequences.

The alternative complex III from Rhodothermus marinus - a prototype of a new family of quinol:electron acceptor oxidoreductases.

The biochemical and genetic search for a bc(1) complex in Rhodothermus marinus was always fruitless; however, a functional equivalent, i.e. having quinol:cytochrome c oxidoreductase activity was characterized. Now, with the sequencing of R. marinus genome, it was possible to assign the N-terminal sequences of several proteins of this complex to its coding genes. The alternative complex III from R. marinus has the same genomic organization of the so-called MFIcc complexes, proposed to be oxidoreductases of the respiratory and photosynthetic electron transfer chains. In this report, we establish undoubtedly the existence of an alternative complex III, a functional substitute of the bc(1) complex, by its identification at both the biochemical and genomic level.

A Rieske ferredoxin typifying a subtype within Rieske proteins: spectroscopic, biochemical and stability studies.

A new subtype of archaeal Rieske ferredoxin (RFd) has been identified in the genome of the thermoacidophilic archaeon Acidianus ambivalens. The gene is inserted in an atypical genomic context in a gene cluster encoding a NiFe hydrogenase. Sequence and phyletic analysis showed that the protein is related to bacterial RFd but not to any of the known archaeal Rieske proteins. The recombinant 14 kDa protein isolated from Escherichia coli behaved as a dimer in solution. It contained approximately 2 Fe/mol and all visible and EPR spectroscopic features typical of Rieske centre-containing proteins. However, its redox potential (+170 mV) was significantly higher than those of canonical RFd. This difference is rationalized in terms of the protein structure environment, as discrete amino acid substitutions in key positions around the metal centre account for the higher potential.

Superoxide anion generation by the cytochrome bc1 complex.

We have measured the rates of superoxide anion generation by cytochrome bc(1) complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc(1) complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc(1) complexes the rate of superoxide anion production was greatest in the absence of bc(1) inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc(1) complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc(1) complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc(1) complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.

A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins.

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.