Mitochondria are the powerhouses of eukaryotic cells as they feed metabolism with its major substrate. Oxidative-phosphorylation relies on the generation, by an electron/proton transfer chain, of an electrochemical transmembrane potential utilized to synthesize ATP. Although these fundamental principles are not a matter of debate, the emerging picture of the respiratory chain diverges from the linear and fluid scheme. Indeed, a growing number of pieces of evidence point to membrane compartments that possibly restrict the diffusion of electron carriers, and to supramolecular assembly of various complexes within various kinds of supercomplexes that modulate the thermodynamic and kinetic properties of the components of the chain. Here, we describe a method that allows the unprecedented time-resolved study of the respiratory chain in intact cells that is aimed at assessing these hypotheses. We show that, in yeast, cytochrome c is not trapped within supercomplexes and encounters no particular restriction to its diffusion which questions the functional relevance of these supramolecular edifices.bioenergetics | electon transfer | respiration | compartmentalization

The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO(2) availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition.

The mutation G143A in the inhibitor binding site of cytochrome b confers a high level of resistance to fungicides targeting the bc 1 complex. The mutation, reported in many plant-pathogenic fungi, has not evolved in fungi that harbor an intron immediately after the codon for G143 in the cytochrome b gene, intron bi2. Using Saccharomyces cerevisiae as a model organism, we show here that a codon change from GGT to GCT, which replaces glycine 143 with alanine, hinders the splicing of bi2 by altering the exon/intron structure needed for efficient intron excision. This lowers the levels of cytochrome b and respiratory growth. We then investigated possible bypass mechanisms that would restore the respiratory fitness of a resistant mutant. Secondary mutations in the mitochondrial genome were found, including a point mutation in bi2 restoring the correct exon/intron structure and the deletion of intron bi2. We also found that overexpression of nuclear genes MRS2 and MRS3, encoding mitochondrial metal ion carriers, partially restores the respiratory growth of the G143A mutant. Interestingly, the MRS3 gene from the plant-pathogenic fungus Botrytis cinerea, overexpressed in an S. cerevisiae G143A mutant, had a similar compensatory effect. These bypass mechanisms identified in yeast could potentially arise in pathogenic fungi.The mitochondrial bc 1 complex is a membrane-bound multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c and couples this electron transfer to the vectorial translocation of protons across the inner mitochondrial membrane. Cytochrome b is the central membraneembedded subunit that forms the ubiquinol binding pockets called Q o and Q i .A number of quinol antagonists are known that inhibit bc 1 complex activity. These are either specific for the Q i site, such as antimycin, or for the Q o site, such as myxothiazol, stigmatellin, and the strobilurins. A range of Q o inhibitor compounds (Q o Is) have been developed as antimicrobial agents and are now widely used in agriculture to control fungal and oomycete plant pathogens. Unfortunately, acquired resistance has rapidly emerged in field populations of the plant pathogens. The cytochrome b mutation G143A plays a central role in the mechanism of resistance. The mutation has been reported in most Q o I-resistant pathogens (see http://www.frac.info/frac /index.htm and references within). G143A causes a high level of resistance (Ͼ100ϫ) in pathogens, which are consequently controlled poorly or not at all by Q o Is. In the model organism Saccharomyces cerevisiae, G143A also dramatically increases resistance to myxothiazol (18,000ϫ) and azoxystrobin (4,000ϫ) (8). G143 is a highly conserved residue located in the Q o pocket, close to the inhibitor binding site. The replacement of glycine with alanine would prevent inhibitor binding through simple steric hindrance while the Q o site remains functional, as observed in the yeast model (8).The cytochrome b gene is encoded by the mitochondrial genome in all eukaryotes. In fungi, lar...