Proton Pumping Capacity Research Articles

Electron Transport Chain Complex II Regulates Steroid Metabolism

SummaryThe first steroidogenic enzyme, cytochrome P450-side-chain-cleavage (SCC), requires electron transport chain (ETC) complexes III and IV to initiate steroid metabolic processes for mammalian survival. ETC complex II, containing succinate dehydrogenase (quinone), acts with the TCA cycle and has no proton pumping capacity. We show that complex II is required for SCC activation through the proton pump, generating an intermediate state for addition of phosphate by succinate. Phosphate anions in the presence of succinate form a stable mitochondrial complex with higher enthalpy (-ΔH) and enhanced activity. Inhibition of succinate action prevents SCC processing at the intermediate state and ablates activity and mitochondrial protein network. This is the first report directly showing that a protein intermediate state is activated by succinate, facilitating the ETC complex II to interact with complexes III and IV for continued mitochondrial metabolic process, suggesting complex II is essential for steroid metabolism regulation.

SUN-219 Electron Transport Chain Complex 2 in Mitochondrial Pregnenolone Synthesis

The mitochondrial P450 family of enzymes (SCC), which require the electron transport chain (ETC) complexes III, IV and V, initiate steroidogenesis by cleaving the sidechain of cholesterol to synthesize steroid hormones, an essential component for mammalian survival. SCC is required for full-term gestation, and aberrant expression may cause pseudohermaphroditism, breast cancer or polycystic ovary syndrome. Complex II or succinate dehydrogenase (quinone) is shared with the TCA cycle and has no proton pumping capacity and no known role in steroid synthesis. We now show that succinate is an intermediate metabolite in the TCA cycle and plays a central role physiologically. Specifically, complex II is required for SCC activation, where the proton pump facilitates an active intermediate state conformation at the matrix, so that in the presence of succinate, ATP can add phosphate. A longer intermediate equilibrium state generates a transient stabilization to enhance the binding of phosphate anions in the presence of succinate anions, resulting in higher enthalpy and activity. An inhibition of the processing at the intermediate state stops phosphate addition and activity. We further describe that phosphate circulation brings the molten globule, an intermediate, to an active folded state. This is the first report showing that an intermediate state activated by succinate facilitates ETC complex II interaction with complexes III and IV for metabolism.

Convergent evolution of unusual complex I homologs with increased proton pumping capacity: energetic and ecological implications.

Respiratory complex I is part of a large family of homologous enzymes that carry out the transfer of electrons between soluble cytoplasmic electron carriers and membrane-bound electron carriers. These complexes are vital bioenergetic enzymes that serve as the entry points into electron transport chains for a wide variety of microbial metabolisms, and electron transfer is coupled to proton translocation. The core complex of this enzyme is made up of 11 protein subunits, with three major proton pumping subunits. Here, we document a large number of modified complex I gene cassettes found in genome sequences from diverse cultured bacteria, shotgun metagenomics, and environmentally derived archaeal fosmids all of which encode a fourth proton pumping subunit. The incorporation of this extra subunit into a functional protein complex is supported by large amino acid insertions in the amphipathic helix that runs the length of the protein complex. Phylogenetic analyses reveal that these modified complexes appear to have arisen independently multiple times in a remarkable case of convergent molecular evolution. From an energetic perspective, we hypothesize that this modification on the canonical complex I architecture allows for the translocation of a fifth proton per reaction cycle-the physiological utility of this modified complex is discussed.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ8,Δ7-isomerases and sterol-Δ14-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H+-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H+-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H+-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H+-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H+-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Cyclic Electron Transfer and Photosynthetic Energy Balance

Cyclic electron flow around photosystem I (CEF1) is thought to balance chloroplast ATP/NADPH output to match downstream demands, but despite decades of work the regulation, pathway, catalytic and proton pumping capacity of the process remain controversial. At least four different pathways have been proposed for PQR, through NAD(P)H:plastoquinone reductase (NDH (1)), the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR (2)), which involved the protein PGR5, through ferredoxin:NADP+ oxidoreductase (FNR, (3)) and through the cytochrome b6f complex (4). In addition, neither the capacity of CEF1, or the system that regulates it are known. We isolated a new class of Arabidopsis mutant, hcef for high CEF1, which shows dramatically higher CEF1. Characterization of these mutants shows that 1) elevated CEF1 flows through NDH rather than other proposed pathways; 2) CEF1 is highly inducible and can achieve quite high rates; 3) CEF1 is critical for maintaining ATP levels under stress; 4) the NDH complex likely pumps protons, like its homolog in the mitochondrion, Complex I. Finally, we demonstrate the energetic importance of CEF1 in stress responses in plants and CO2 pumping in algae.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H(+)/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

Differences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C3/CAM intermediate plantKalanchoe blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H+-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V0 domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H+-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.

Proton pumping of mitochondrial complex I: differential activation by analogs of ubiquinone.

As part of the ongoing studies aimed at elucidating the mechanism of the energy conserving function of mitochondrial complex I, NADH: ubiquinone (Q) reductase, we have investigated how short-chain Q analogs activate the proton pumping function of this complex. Using a pH-sensitive fluorescent dye we have monitored both the extent and initial velocity of proton pumping of complex I in submitochondrial particles. The results are consistent with two sites of interaction of Q analogs with complex I, each having different proton pumping capacity. One is the physiological site which leads to a rapid proton pumping and a stoichiometric consumption of NADH associated with the reduction of the most hydrophobic Q analogs. Of these, heptyl-Q appears to be the most efficient substrate in the assay of proton pumping. Q analogs with a short-chain of less than six carbons interact with a second site which drives a slow proton pumping activity associated with NADH oxidation that is overstoichiometric to the reduced quinone acceptor. This activity is also nonphysiological, since hydrophilic Q analogs show little or no respiratory control ratio of their NADH:Q reductase activity, contrary to hydrophobic Q analogs.

Effect of cholate on H(+)-ATPase and other proteins of dog renal brush-border membrane.

A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H(+)-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H(+)-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits ot the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H(+)-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H(+)-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but gamma-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H(+)-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H(+)-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H(+)-ATPase of proximal tubules.

Sodium Chloride Compartmentation in Leaf Vacuoles of the Halophyte Suaedamaritima(L.) Dum. and its Relation to Tonoplast Permeability

Single channel properties, whole vacuole currents and proton pumping capacity were investigated in the intact vacuoles and membrane patches of leaf tonoplast from the halophyte Suaeda maritima. ATP-dependent proton pumping capacity was similar to non-halophytes whether the plants were or were not grown with added sodium chloride (200 mM). The most abundant ion channel was inward rectifying and had a single channel conductance of 58 pS in symmetrical KCl solutions (100 mM) to 170 pS using physiological conditions (50/150 mM KCl/NaCl cytoplasmic side, 50/450 mM KCl/NaCl vacuolar side). The channel showed all the characteristics of the SV type channel described in many other species. In the open state these channels caused tonoplast conductances in excess of 0.5 nS m2– but conductances were much lower using physiological ion concentrations and membrane potentials. In spite of the poor selectivity and the potentially large tonoplast conductance it is calculated that compartmentation of NaCl in leaf vacuoles can be sustained by about 30% of ATP-dependent proton pumping capacity. The results do not indicate any special adaptation of the tonoplast ion channels in the halophyte.

Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani.

ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units.

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.