Cytochrome c Oxidase at Full Thrust: Regulation and Biological Consequences to Flying Insects.

Flight dispersal represents a key aspect of the evolutionary and ecological success of insects, allowing escape from predators, mating, and colonization of new niches. The huge energy demand posed by flight activity is essentially met by oxidative phosphorylation (OXPHOS) in flight muscle mitochondria. In insects, mitochondrial ATP supply and oxidant production are regulated by several factors, including the energy demand exerted by changes in adenylate balance. Indeed, adenylate directly regulates OXPHOS by targeting both chemiosmotic ATP production and the activities of specific mitochondrial enzymes. In several organisms, cytochrome c oxidase (COX) is regulated at transcriptional, post-translational, and allosteric levels, impacting mitochondrial energy metabolism, and redox balance. This review will present the concepts on how COX function contributes to flying insect biology, focusing on the existing examples in the literature where its structure and activity are regulated not only by physiological and environmental factors but also how changes in its activity impacts insect biology. We also performed in silico sequence analyses and determined the structure models of three COX subunits (IV, VIa, and VIc) from different insect species to compare with mammalian orthologs. We observed that the sequences and structure models of COXIV, COXVIa, and COXVIc were quite similar to their mammalian counterparts. Remarkably, specific substitutions to phosphomimetic amino acids at critical phosphorylation sites emerge as hallmarks on insect COX sequences, suggesting a new regulatory mechanism of COX activity. Therefore, by providing a physiological and bioenergetic framework of COX regulation in such metabolically extreme models, we hope to expand the knowledge of this critical enzyme complex and the potential consequences for insect dispersal.

Mitochondrial glycerol phosphate oxidation is modulated by adenylates through allosteric regulation of cytochrome c oxidase activity in mosquito flight muscle.

The huge energy demand posed by insect flight activity is met by an efficient oxidative phosphorylation process that takes place within flight muscle mitochondria. In the major arbovirus vector Aedes aegypti, mitochondrial oxidation of pyruvate, proline and glycerol 3-phosphate (G3P) represent the major energy sources of ATP to sustain flight muscle energy demand. Although adenylates exert critical regulatory effects on several mitochondrial enzyme activities, the potential consequences of altered adenylate levels to G3P oxidation remains to be determined. Here, we report that mitochondrial G3P oxidation is controlled by adenylates through allosteric regulation of cytochrome c oxidase (COX) activity in A. aegypti flight muscle. We observed that ADP significantly activated respiratory rates linked to G3P oxidation, in a protonmotive force-independent manner. Kinetic analyses revealed that ADP activates respiration through a slightly cooperative mechanism. Despite adenylates caused no effects on G3P-cytochrome c oxidoreductase activity, COX activity was allosterically activated by ADP. Conversely, ATP exerted powerful inhibitory effects on respiratory rates linked to G3P oxidation and on COX activity. We also observed that high energy phosphate recycling mechanisms did not contribute to the regulatory effects of adenylates on COX activity or G3P oxidation. We conclude that mitochondrial G3P oxidation in A. aegypti flight muscle is regulated by adenylates through the allosteric modulation of COX activity, underscoring the bioenergetic relevance of this novel mechanism and the potential consequences for mosquito dispersal.

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric.

Mitochondrial F(1)F(O)-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F(1) sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0-9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5.

Pathogenic effector T cell enrichment overcomes regulatory T cell control and generates autoimmune gastritis.

Regulatory T cells (Treg) deficiency leads to a severe, systemic, and lethal disease, as showed in immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome patients, and scurfy mouse. Postneonatal thymectomy autoimmune gastritis has also been attributed to the absence of Tregs. In this case however, disease is mild, organ-specific, and, more important, it is not an obligatory outcome. We addressed this paradox comparing T cell compartments in gastritis-susceptible and resistant animals. We found that neonatal thymectomy-induced gastritis is not caused by the absence of Tregs. Instead of this, it is the presence of gastritogenic T cell clones that determines susceptibility to disease. The expansion of such clones under lymphopenic conditions results in a reduced Treg:effector T cell ratio that is not enough to control gastritis development. Finally, the presence of gastritogenic clones is determined by the amount of gastric Ag expressed in the neonatal thymus, emphasizing the importance of effector repertoire variability, present even in genetically identical subjects, to organ-specific autoimmune disease susceptibility.

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid.

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.

Effects of gamma-irradiation on the membrane ATPases of human erythrocytes from transfusional blood concentrates.

Irradiation of blood derivatives is employed in blood banks to avoid transfusion-associated graft-vs-host disease. As irradiation can damage membranes and membrane proteins by generation of reactive oxygen species, we investigated whether the membrane permeability, Na(+),K(+)-ATPase, and Ca(2+)-ATPase from red blood cell plasma membranes were altered by gamma-irradiation. Whole blood was collected from healthy donors and concentrated to 90% cell fraction. Within 24 h of collection, blood concentrates were irradiated with 25 Gy of gamma-radiation. At days 1, 7, 14, and 28 post-irradiation, fractions were removed and centrifuged. Na(+),K(+)-ATPase and Ca(2+)-ATPase activities from ghost membranes were assessed by gamma-(32)P-ATP hydrolysis. The Na(+),K(+)-ATPase was not immediately affected by irradiation, but it was inhibited by 40% by day 14 and until day 28. The Ca(2+)-ATPase was unaltered by irradiation. The rate and the maximal (45)Ca(2+) uptake from re-sealed inside-out vesicles were reduced, and the passive efflux of (45)Ca(2+) was increased. Thus, irradiation of blood concentrates increased the plasma membrane permeability to monovalent and divalent cations and would change ion homeostasis and cell function. We recommend the use of irradiated blood within a period shorter than 14 days after irradiation.

Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system.

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).

Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.

Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium.

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.

High hydrostatic pressure perturbs the interactions between CF(0)F(1) subunits and induces a dual effect on activity.

Chloroplast ATP-synthase is an H(+)/ATP-driven rotary motor in which a hydrophobic multi-subunit assemblage rotates within a hydrophilic stator, and subunit interactions dictate alternate-site catalysis. To explore the relevance of these interactions for catalysis we use hydrostatic pressure to induce conformational changes and/or subunit dissociation, and the resulting changes in the ATPase activity and oligomer structure are evaluated. Under moderate hydrostatic pressure (up to 60-80 MPa), ATPase activity is increased by 1.5-fold. This is not related to an increase in the affinity for ATP, but seems to correlate with an enhanced turnover induced by pressure, and an activation volume for the ATPase reaction of -23.7 ml/mol. Higher pressure (up to 200 MPa) leads to dissociation of the enzyme, as shown by enzyme inactivation, increased binding of 8-anilinonaphthalene-1-sulfonate (ANS) to hydrophobic regions, and labeling of specific Cys residues on the beta and alpha subunits by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-4-diamine (IAEDANS). Compression-decompression cycles (between 0.1 and 200 MPa) inactivate CF(0)F(1) in a concentration-dependent manner, although after decompression no enzyme subunit is retained on a Sephadex-G-50 centrifuge column or is further labeled by IAEDANS. It is proposed that moderate hydrostatic pressures induce elastic compression of CF(0)F(1), leading to enhanced turnover. High pressure dissociation impairs the contacts needed for rotational catalysis.

Conformational changes of the nucleotide site of the plasma membrane Ca2+-ATPase probed by fluorescence quenching.

Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.

3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.