Inhibition of the mitochondrial respiratory chain by alkylhydroxynaphthoquines: Reversal on discharge of the energized state

Inhibition of mitochondrial respiration by alkylhydroxynaphthoquinones may be reversed by addition of a variety of uncouplers including substituted phenols, carbonyl cyanide phenylhydrazones, divalent cations and univalent cations in the presence of ionophoretic antibiotics. A likely explanation for such reversibility is the requirement that the anionic inhibitor be transported to a site of action within the mitochondrion. Support for this view includes (1) failure to obtain reversal of inhibition with submitochondrial particles, (2) release of inhibition by a competing anion, succinate, (3) augmentation of inhibition when a divalent cation is taken up, (4) the chemical diversity of uncouplers that release inhibition and (5) inhibiton by uncoupling compounds of the uptake of labeled alkylhydroxynaphthoquinones. It is suggested that a similar explanation may apply to two other inhibitors of the cytochrome b–c region, antimycin and alkylhydroxyquinoline-N-oxides.     

Supersizing the mitochondrial respiratory chain

The complexes of the mitochondrial respiratory chain assemble into higher-order structures called supercomplexes or respirasomes that are thought to be important in channeling electron flow and controlling ROS production. A number of recent papers identify the first protein factors necessary for supercomplex assembly and stability.     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.     

Succinate-ubiquinone reductase site of the respiratory chain

Data on succinate-ubiquinone reductase are critically reviewed. The structural and catalytic properties of succinate dehydrogenase and succinate-ubiquinone reductase are compared. The redox components, active centers and proteins involved in the enzyme interaction with ubiquinone are described. Some structural and kinetic features of the succinate-ubiquinone reductase as the respiratory chain component and feasible mechanisms of regulation of the succinate-ubiquinone reductase activity are discussed.     

A three-cycle mechanism of the respiratory chain

A scheme of the respiratory chain is presented, according to which three delta mu H-generators (complexes I, III and IV) provide for the transmembrane transport of ten H+ ions per atom of adsorbed O2. It is assumed that all the three delta mu H-generators operate in accordance with the same mechanism, namely, they translocate electrons at a distance averaging 1/2 of membrane width, whereas protons moving in the opposite direction pass the other halfwidth across the membrane. A redox cycle functions in each of the three sites of the energy coupling mechanism: the flavin (F) cycle in complex I, the Q-cycle in complex III and the O-cycle in complex IV. These cycles are interconnected by mobile carriers: the F- and Q-cycles by ubiquinone and the Q- and O-cycles by cytochrome c.     

The development of the respiratory chain of Saccharomyces carlsbergensis during respiratory adaptation

1. Subcellular fractionation of sphaeroplasts produced at different stages during the first 4h of respiratory adaptation of anaerobically grown glucose-de-repressed Saccharomyces carlsbergensis gave mitochondrial fractions that contained all the detectable c- and a-type cytochromes. 2. The rates of cytochrome formation were studied; individual cytochromes were produced at different rates so as to give respiratory chains having widely differing cytochrome ratios. A CO-reacting haemoprotein other than cytochrome a(3) also increased throughout 8h of respiratory adaptation. 3. Even after short periods of aeration, organisms contained mitochondria in which cytochrome-cytochrome interactions and the reaction of cytochrome a(3) with O(2) proceeded at rates almost as fast as in organelles from aerobically grown cells. 4. The technique of flow-flash photolysis enabled kinetic resolution of the reoxidation of cytochromes a(3) and a to be achieved and their individual contributions to extinction changes in the Soret region were assessed. The ratio cytochrome a(3)/cytochrome a increased over the early stages of adaptation.     

Characterization of the respiratory chain of Helicobacter pylori

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.     

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.     

Instability of the tetrathionate respiratory chain ofCitrobacter freundii

The formate—tetrathionate reductase redox pathway ofCitrobacter freundii is associated with the cytoplasmic membrane fraction. A high concentration of phosphate in the buffer used for cell disintegration assists in the preparing of membrane particles capable of efficient tetrathionate respiration. A part of this effect at least may be attributed to the high ionic strength of the buffer.     

Reversal of depressed miduterine arterial flow during hyperthermia-induced respiratory alkalosis

The influence of ambient and arterial PCO2 on miduterine arterial flow of pregnant sheep acutely exposed to hot environments was investigated. Five mixed-breed ewes between 120 and 130 days of gestation were subjected to hot environments (increasing from thermoneutral 23 to 40 degrees C), and arterial blood pH, PCO2, and PO2 were determined at 5-min intervals. Respiratory rate, heart rate, rectal temperature, blood pressure, and miduterine arterial flow were continuously monitored prior to and during elevation of ambient air temperature. When miduterine arterial flow had decreased to 50% of thermoneutral control levels, ambient air CO2 was increased to 2.5%. Elevated ambient inspired CO2 caused a reversal in arterial pH and PCO2 to near thermoneutral levels. Miduterine arterial flow increased to 77% of the control levels following the elevated ambient PCO2 period. Respiratory rate also decreased when ambient CO2 was increased but remained 136% greater than the thermoneutral control level. All other parameters remained near their heat stress (40 degrees C) level during the elevation of ambient CO2. These data indicate that heat-stress-induced depression of miduterine arterial flow is vasoactively regulated, and cause-effect related to both arterial pH and PCO2, and thermoregulatory shunting of blood to heat-dissipating surfaces.     

Cardiolipin stabilizes respiratory chain supercomplexes

Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.     

On the role of ubiquinone in the respiratory chain

(1) The V₁ (substrate-Q oxidoreductase activity) and V₂ (QH₂ oxidase activity) for the oxidation of substrates by submitochondrial particles have been measured by using heptylhydroxyquinoline N-oxide (HQNO) as inhibitor of V₂. (2) Partial destruction of the Rieske Fe-S cluster by treatment with BAL (2,3-dimercaptopropanol)+O₂ has the same effect on the QH₂ oxidase activity as partial saturation of the antimycin-binding site with HQNO. (3) The extent of the rapid reduction of cytochrome b in the presence of excess antimycin is proportional to the percentage of intact Rieske Fe-S cluster. (4) The measured rate of oxidation of endogenous ubiquinol (V₂) by submitochondrial particles is dependent on the substrate used to reduce ubiquinone, especially at low levels of ubiquinone. (5) Pool-function kinetics in the oxidation of substrate, found both in the presence and absence of free ubiquinone, are due both to the pool of free ubiquinone and to direct collision between Q-loaded Q-reducing and -oxidizing enzymes. At infinite Q content only the former mechanism is operative; at low Q content only the latter. (6) Duroquinone can be reduced directly by NADH dehydrogenase without mediation of ubiquinone, but duroquinol cannot be oxidized in the absence of ubiquinone. On the other hand, the reduction of cytochrome b by duroquinol does not require the presence of ubiquinone. (7) It is suggested that the need for ubiquinone for the oxidation of duroquinol is due to the requirement of ubisemiquinone for the oxidation of cytochrome b, duroquinol not being able to form a stabilized semiquinone.     

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.