The mitochondrial F-ATPases make about 90% of cellular ATP. They are multi-protein assemblies with a membrane extrinsic catalytic domain attached to a membrane embedded sector. They operate by a mechanical rotary mechanism powered by an electro-chemical gradient, generated across the inner mitochondrial membrane by respiration. A detailed molecular description has been provided by X-ray crystallographic studies and "single molecule" observations of the mechanism of the F1 catalytic domain. Details are known also of the architecture of the peripheral stalk of part of the stator and the membrane embedded region of the rotor. However, knowledge of the detailed structure of the rest of the membrane domain, and the detailed mechanism of generation of rotation is lacking. Recently, studies of the intact mitochondrial F-ATPases, determined by cryo-electron microscopy (cryo-em), have provided structural information at intermediate levels of resolution. Whilst these structures have given insights into the mechanism of generation of rotation, the information required for a molecular understanding of this mechanism is still lacking. Moreover, the locations and roles of six supernumerary membrane subunits are unclear. Some of them are likely to be involved in the formation of dimers of the enzyme which line the edges of mitochondrial cristae. Therefore, in this thesis, a procedure is described for the purification of dimers of the bovine and yeast F-ATPases. The structure of the bovine dimer has been determined by cryo-em at a resolution of ca. 6.9 Angstrom. This structure confirms features concerning the trans-membrane spans of the a-, A6L- and b-subunits observed in the monomeric complex. In addition, the single trans-membrane a-helix of the f-subunit has been located, and the subunit appears to mediate dimer formation. The structure of A6L has been extended, and the a-helices of subunits e- and g- have been located. Another novel feature has been assigned to the DAPIT subunit, and may provide links between dimers in forming larger oligomers. Further improvement in the resolution of the structure is hampered by the extreme conformational heterogeneity of the F-ATPase. To this end, the simpler Fo membrane domain has been isolated and characterized initially by electron microscopy in negative stain.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of 17 bioflavonoid compounds on purified F1 or membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC50∼0.07mM)>silymarin (IC50∼0.11mM)>baicalein (IC50∼0.29mM)>silibinin (IC50∼0.34mM)>rimantadin (IC50∼2.0mM)>amantidin (IC50∼2.5mM)>epicatechin (IC50∼4.0mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F1Fo membrane preparations and isolated purified F1. ATPase and growth assays suggested that the bioflavonoid compounds used in this study inhibited F1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo-ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (∼96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of ∼13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC50 ∼3.25 μM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase (∼20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F1Fo membrane preparations as well as in isolated purified F1. Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F1-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase.

Venom peptides are known to have strong antimicrobial activity and anticancer properties. King cobra cathelicidin or OH-CATH (KF-34), banded krait cathelicidin (BF-30), wolf spider lycotoxin I (IL-25), and wolf spider lycotoxin II (KE-27) venom peptides were found to strongly inhibit Escherichia coli membrane bound F1Fo ATP synthase. The potent inhibition of wild-type E. coli in comparison to the partial inhibition of null E. coli by KF-34, BF-30, Il-25, or KE-27 clearly links the bactericidal properties of these venom peptides to the binding and inhibition of ATP synthase along with the possibility of other inhibitory targets. The four venom peptides KF-34, BF-30, IL-25, and KE-27, caused ≥85% inhibition of wild-type membrane bound E.coli ATP synthase. Venom peptide induced inhibition of ATP synthase and the strong abrogation of wild-type E. coli cell growth in the presence of venom peptides demonstrates that ATP synthase is a potent membrane bound molecular target for venom peptides. Furthermore, the process of inhibition was found to be fully reversible.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-β-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC50 ∼14 μM) followed by quercetin (IC50 ∼33 μM), quercetin-3-β-d glucoside (IC50 ∼71 μM), resveratrol (IC50 ∼94 μM), quercitrin (IC50 ∼120 μM). Inhibition was identical in both F1Fo membrane preparations as well as in isolated purified F1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.

The role of αPhe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.

1. Aufgrund der Resultate mit der Mutante MM10, die trotz Disulfidbrücke noch unverminderte Aktivität und Rotation zeigte, wurde der Frage nachgegangen, ob der EF1-Komplex in der Lage ist, die Rotation durch einen Bruch der alpha-helikalen Struktur von gamma oder durch Rotation um eine Einfachbindung der Disulfidbrücke aufrechtzuerhalten. Quervernetzungen vom Hexagon mit gamma in der Mitte und am unteren Ende konnten das Enzym blockieren. Von den vier betrachteten Mutanten KG11, MM26, MM25 und MM24 fiel der MM26 bereits nach der Isolierung raus. Der MM25 wies nicht mehr als 70% Quervernetzung auf. Bei dem KG11 und dem MM24 konnte jedoch eine 99%-ige Quervernetzung nachgewiesen werden. Mit diesen zwei Cystein-Doppelmutanten wurden weitere Quervernetzungen gefunden, die ebenso wie der MM10 aktiv nach Oxidation sind, aber tiefer im Enzym liegen und die ATP-Hydrolyse trotz Blockade durch Quervernetzung aufrecht erhalten. Es konnte gezeigt werden, dass eine Rotation um die Einzelbindungen innerhalb der Disulfidbrücke unwahrscheinlich ist, und daher die Aktivität des quervernetzten Enzyms nur durch eine Aufwindung der gamma-Helix erklärt werden kann. 2. Die Voraussetzung für ein EFOF1-Kostrukt zum optischen Nachweis der Relativrotation unter Synthesebedingungen war der Einbau von zwei verschiedenen Tags zur spezifischen Bindung. Von den vier Mutanten SE3, SE4, SW7 und WH1 zeigten die beiden SE-Mutanten keine Stabilität bei der Isolierung im Bezug auf die Kopplung zwischen FO- und F1-Teil. Mit dem SW7-EFOF1 wurde eine Mutante gefunden, die mit einer guten Aktivitäts- und Rotationsausbeute nach einer Aufreinigung mittels Streptactin-Affinitätchromatographie durch ihre Stabilität als Ausgangspunkt für das Rotationsexperiment unter ATP-Synthese dienen kann. Der WH1, dessen atp-Operon dem des SW7 gleicht, brachte trotz seines veränderten Vektorursprungs keine Verbesserung.