S1.20 Divalent metal binding to bovine heart F1 ATPase: An FT-ESEEM study

The divalent metal binding sites of beef heart mitochondria F1ATPase were studied by FT-ESEEM spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II) and maintains the enzyme catalytic activity. Purified F1ATPase still containing three endogenous tightly bound nucleotides, named MF1(1,2), was obtained under mild conditions, whereas a harsher treatment gave a fully nucleotide depleted enzyme, named MF1(0,0). When MF1(1,2) was loaded with Mn(II) in 1:0.8 ratio, the spectrum showed evidence of a nitrogen interacting with the metal, while this interaction was not present in the spectrum of MF1(0,0) loaded with Mn(II) in the same ratio. However, when MF1(0,0) was loaded with 2.4 Mn(II), the spectrum showed metal-nitrogen interaction resembling that of MF1(1,2) loaded with Mn(II) in 1:0.8 ratio. When MF1(1,2) was loaded with 2.4 Mn(II) the metal-nitrogen interaction signal remained and a phosphorous coordination to the metal was also evident, indicating a binding of Mn2+ to a site containing a tightly bound nucleotide but metal free. These results strongly support the role of the metal alone in structuring the catalytic sites of the enzyme while ESEEM technique appears to be a sensitive and suitable spectroscopic method for conformational studies of MF1 with the advantage of using proteins in frozen solution

Structural and functional properties of plant mitochondrial F-ATP synthase

The mitochondrial F-ATP synthase is responsible for coupling the transmembrane proton gradient, generated through the inner membrane by the electron transport chain, to the synthesis of ATP. This enzyme shares a basic architecture with the prokaryotic and chloroplast ones, since it is composed of a catalytic head (F1), located in the mitochondrial matrix, a membrane-bound part (FO), together with a central and a peripheral stalk. In this review we compare the structural and functional properties of F-ATP synthase in plant mitochondria with those of yeast and mammals. We also present the physiological impact of the alteration of F-ATP synthase in plants, with a special regard to its involvement in cytoplasmic male sterility. Furthermore, we show the involvement of this enzyme in plant stress responses. Finally, we discuss the role of F-ATP synthase in shaping the curvature of the mitochondrial inner membrane and in permeability transition pore formation

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different

Partial uncoupling, or inhibition of electron transport rate, have equivalent effects on the relationship between the rate of ATP synthesis and proton-motive force in submitochondrial particles

AbstractThe rates of electron transport and of ATP synthesis have been measured in bovine heart Mg-ATP submito-chondrial particles oxidising succinate under conditions of partial attenuation of the proton-motive force by malonate or FCCP. This paper reports evidence that the relationship between the rate of ATP synthesis and the magnitude of the proton motive force is independent of the mode by which the decrease of the proton motive force is achieved

Effects of Fe(III) binding to the nucleotide-independent site of F1-ATPase: enzyme thermostability and response to activating anions

AbstractMitochondrial F1-ATPase was induced in different conformations by binding of specific ligands, such as nucleotides. Then, Fourier transform infrared spectroscopy (FT-IR) and kinetic analyses were run to evaluate the structural and functional effects of Fe(III) binding to the nucleotide-independent site. Binding of one equivalent of Fe(III) induced a localised stabilising effect on the F1-ATPase structure destabilised by a high concentration of NaCl, through rearrangements of the ionic network essential for the maintenance of enzyme tertiary and/or quaternary structure. Concomitantly, a lower response of ATPase activity to activating anions was observed. Both FT-IR and kinetic data were in accordance with the hypothesis of the Fe(III) site location near one of the catalytic sites, i.e. at the α/β subunit interface

Effect of ultrasound treatment, oil addition and storage time on lycopene stability and in vitro bioaccessibility of tomato pulp

This study was performed to investigate the influence of ultrasound processing on tomato pulp containing no sunflower oil, or increasing amounts (i.e. 2.5%, 5% and 10%), on lycopene concentration and in vitro bioaccessibility at time zero and during storage at 5 \ub0C. Results confirmed previous findings in that ultrasonication was responsible for cell breakage and subsequent lycopene release in a highly viscous matrix. Neither the ultrasound process nor oil addition affected lycopene concentration. A decrease of approximately 35% lycopene content occurred at storage times longer than 15 days, due to isomerisation and oxidation reactions. No differences in lycopene in vitro bioaccessibility were found between the untreated and ultrasonically treated samples; this parameter decreased as a consequence of oil addition. Losses of lycopene in vitro bioaccessibility ranging between 50% and 80% occurred in the untreated and ultrasonically treated tomato pulps with and without oil during storage, mainly due to carotenoid degradation

Nanoemulsions as delivery systems of hydrophobic silybin from silymarin extract: Effect of oil type on silybin solubility, invitro bioaccessibility and stability

The potential of nanoemulsion delivery systems to carry silybin from silymarin extract was studied. To this purpose, sunflower oil, extra virgin olive oil and castor oil were used to prepare silymarin loaded nanoemulsions. The effect of oil type on the silybin solubility and i. n vitro bioaccessibility was evaluated. Moreover, the changes in particle size, silybin concentration, oxygen consumption and hydroperoxide concentration were studied in nanoemulsions during storage at 20\ub0C. Results showed that silybin can be successfully incorporated into physically stable nanoemulsions prepared with the different oils. The oil type slightly influenced the silybin invitro bioaccessibility, while it affected the nanoemulsion particle size as well as silybin stability during storage. In particular, silybin underwent degradation, showing lower stability in extra virgin oil and sunflower oil than in castor oil. Results also showed that the presence of the silymarin extract containing silybin did not affect the oxidation kinetics of the carrier oils. \ua9 2015 Elsevier Ltd

Channel formation by yeast F-ATP synthase and the role of dimerization in the mitochondrial permeability transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca(2+)-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca(2+) ionophore ETH129, which allows electrophoretic Ca(2+) uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains \u394TIM11 and \u394ATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ

Properties of the permeability transition of pea stem mitochondria

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by MgADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel