The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc1 complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.

A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.

Mgr2 is a new component of the mitochondrial presequence translocase required for efficient coupling of the TIM23 core complex to Tim21, respiratory chain complexes, and the translocase of the outer mitochondrial membrane.

We previously proposed that the dimeric cytochrome bc 1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc 1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289 -22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc 1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10 -16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c 1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wildtype dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.The cytochrome bc 1 complex is a multisubunit enzyme that generates a transmembrane protonmotive force by transferring electrons from ubiquinol to cytochrome c. Energy conservation in this enzyme complex is ensured by the oxidation of ubiquinol and the reduction of ubiquinone at active sites located on opposite sides of the membrane, as described by the protonmotive Q-cycle (1). High resolution structures have shown that the bc 1 complex is a dimer composed of two copies of cytochrome b, the Rieske iron-sulfur protein, and cytochrome c 1 , which are the only polypeptides present in some bacterial enzymes (2), in addition to six to eight additional subunits present exclusively in each monomer of the eukaryotic complex (3-5).The functional relevance of this dimeric arrangement has been supported by an extensive body of kinetic evidence (for review, see Ref. 6), which includes the key observations that only one ubiquinol oxidation site, or center P, is active when the two ubiquinone reduction, or center N, sites are occupied by inhibitors (7), that electrons rapidly equilibrate between the cytochrome b subunits (8, 9), and that there is conformational communication between center P and center N sites (10). We have proposed that this half-of-the sites activity at center P also exists under normal steady-state conditions in the absence of inhibitors and that this mechanism minimizes the leakage of electrons to oxygen under conditions that would favor the accumulation of electrons in the cytochrome b hemes (8, 11). Evidence consistent with this...

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.