Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent

Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH.     

Decylubiquinol impedes mitochondrial respiratory chain complex I activity

In this study, indirect immunofluorescence labeling was used to examine the cellular dynamic distribution of Thr11 phosphorylated H3 at mitosis in MCF-7 cells. The Thr11 phosphorylation was observed beginning at prophase at centromeres. Upon progression of mitosis, fluorescence signal was enhanced in the central region of the metaphase plate and maintained till anaphase at centromeres. During telophase, the fluorescent signal of Thr11 phosphorylated H3 disappears from centromeres, but the signal appears again at the midbody during cytokinesis, which suggests that the modified histones may take part in the formation of the midbody and play a crucial role in cytokinesis. Chromatin immunoprecipitation (ChIP) was used to confirm that Thr11 phosphorylated H3 is specifically associated with centromere DNA at prophase to metaphase, which is coincident with the results observed by immunofluorescence. In conclusion, there was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and stages of chromatin condensation. The timing of Thr11 phosphorylation and dephosphorylation in mitosis were similar to that reported for Ser10 phosphorylation of H3. The Thr11 phosphorylated H3 localized at centromeres during mitosis, which was different from the Ser10 phosphorylated H3 localized at telomere regions and Thr3 phosphorylated H3 localized along the chromosome arms. The results suggest that the Thr11 phosphorylation of histone H3 may play a specific role which was different from Ser10 and Thr3 phosphorylation in mitosis.     

Tissue specific defect of complex I of the mitochondrial respiratory chain

Deficiency of complex I is one of the most commonly reported defects of the mitochondrial respiratory chain in man. Clinical evidence of tissue specific expression of complex I deficiency has not previously been confirmed biochemically. We report here slow oxidation of NAD+-linked substrates, low activity of complex I and low amounts of immunoreactive complex I peptides in skeletal muscle mitochondria from a patient with muscle weakness and lactic acidosis. In liver mitochondria complex I activity was normal and all the immunoreactive subunits of complex I were present in normal amounts.     

Effects of mitochondrial complex III disruption in the respiratory chain of Neurospora crassa

In mitochondria from most organisms, including Neurospora crassa , dimeric complex III was found associated with complex I. Additional association of complex IV with this core structure leads to the formation of a respirasome. It was recently described for bacteria and mammals that complex III is needed for the assembly/stability of complex I. To elucidate the role of complex III in the organization of the respiratory chain of N. crassa , we analysed strains devoid of either the Rieske iron-sulphur or the COREII polypeptide subunits. The mutants display reduced growth, are female sterile and lack active complex III. The supramolecular organization of the oxidative phosphorylation system was characterized by electrophoretic analyses and the efficiency of the respiratory chain analysed by oxygen consumption measurements. The results obtained indicate that absence of complex III activity is not associated with the absence of complex I or complex IV, and leads to the induction of alternative oxidase. Complex III mutant mitochondria are devoid of respirasomes but contain significant amounts of dimeric complex I (I₂) and of the supercomplex I₁IV₁. Moreso, for the first time the alternative oxidase was found associated with dimeric complex IV and with supercomplex I₁IV₁.     

Effect of cross-linking agents on electron transport and proton transfer in the mitochondrial respiratory chain

The effect of protein cross-linkage on proton translocation and electron transport in mitochondria respiratory chain was studied. Dimethylsuberimidate (1 mM) or dicyclohexyl carbodiimide (50 g/ml) inhibit proton translocation with concomitant stimulation of respiration. It is concluded that the definite level of dynamic mobility of proteins is needed for proton translocation.     

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.     

Tracing the trail of protons through complex I of the mitochondrial respiratory chain

Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes--the respiratory chain--that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

Gluing the respiratory chain together. Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane

Cytochrome bc(1) complex (complex III) and cytochrome c oxidase complex (complex IV) are multisubunit homodimers that are essential components of the mitochondrial respiratory chain. Complexes III and IV associate to form a supercomplex that can be displayed using blue native polyacrylamide gel electrophoresis. Both homodimeric complexes contain tightly associated cardiolipin (CL) required for function. We report here that in a crd1Delta strain of yeast (null in expression of CL synthase) approximately 90% of complexes III and IV were observed as individual homodimers; only the supercomplex was observed with CRD1 wild type cells. Introduction of a plasmid born copy of the CRD1 gene under exogenous regulation by doxycycline made possible controlled variation in the in vivo CL levels. At an intermediate level of CL, a mixture of individual homodimers (30%) and supercomplex (70%) was observed. These results strongly indicate that CL plays a central role in higher order organization of components of the respiratory chain of mitochondria.     

Kinetics of electron transfer through the respiratory chain

We show that the rate at which electrons pass through the respiratory chain in mitochondria and respiring prokaryotic cells is described by the product of three terms, one describing electron donation, one acceptance, and a third, the thermodynamic drive. We apply the theory of nonequilibrium thermodynamics in the context of the chemiosmotic model of proton translocation and energy conservation. This approach leads to a closed-form expression that predicts steady-state electron flux as a function of chemical conditions and the proton motive force across the mitochondrial inner membrane or prokaryotic cytoplasmic membrane. The rate expression, derived considering reverse and forward electron flow, is the first to account for both thermodynamic and kinetic controls on the respiration rate. The expression can be simplified under specific conditions to give rate laws of various forms familiar in cellular physiology and microbial ecology. The expression explains the nonlinear dependence of flux on electrical potential gradient, its hyperbolic dependence on substrate concentration, and the inhibiting effects of reaction products. It provides a theoretical basis for investigating life under unusual conditions, such as microbial respiration in alkaline waters.     

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol.     

Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I

The present study shows that rat liver and brain mitochondrial nitric oxide synthase (mtNOS) are functionally associated with mitochondrial respiratory chain complex I. When complex I is activated, mtNOS exerts high activity and generates nitric oxide, whereas inactivation of complex I leads mtNOS to abandon its NOS activity. Functional association of mtNOS with complex I is potentially important in regulating mtNOS activity and mitochondrial functions.     

Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts

Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.     

Inactivation of mitochondrial respiratory chain complex I leads mitochondrial nitric oxide synthase to become pro-oxidative

We recently demonstrated that mitochondrial nitric oxide synthase (mtNOS) functionally couples with mitochondrial respiratory chain complex I to produce nitric oxide [M.S. Parihar, R.R. Nazarewicz, E. Kincaid, U. Bringold, P. Ghafourifar, Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I, Biochem. Biophys. Res. Commun. 366 (2008) 23-28] [1]. The present report shows that inactivation of complex I leads mtNOS to become pro-oxidative. Our findings suggest a crucial role for mtNOS in oxidative stress caused by mitochondrial complex I inactivation.     

Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.

Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.