F 1 -ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120°power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35°from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85°to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85°of rotation, consistent with a transition from F 1 structures 2HLD1 and 1H8E (Protein Data Bank).

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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 seventeen 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) > rimantadine (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 isolated purified F1. ATPase and growth assays suggested that the bioflavonoids 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.

. ) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response.

Residues responsible for phosphate binding in F 1 F 0 -ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved ␤Arg-246 in a suitable position to bind phosphate. Mutations ␤R246Q, ␤R246K, and ␤R246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F 1 by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. 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 ϳ50%, although reaction still occurred at residue ␤Tyr-297, proximal to ␤Arg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ␤E (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the ␤E catalytic site of E. coli F 1 and that ␤Arg-246 is an important phosphate-binding residue.ATP synthesis from ADP and P i in oxidative and photophosphorylation occurs in the catalytic sites of F 1 F 0 -ATP synthase. This membrane enzyme uses the energy of an ion gradient (usually protons) to drive rotation of a "rotor" composed of subunits ␥⑀c n (where n can vary from 10 to 14 in different organisms), the rotor serving as a transmission device which upon rotation drives the chemical synthesis of ATP sequentially at three catalytic sites. Catalytic sites are located at ␣/␤ interfaces of the ␣ 3 ␤ 3 subunit hexagon, which are immobilized during rotation by the "stator," consisting of subunits ␦b 2 . The proton motor is composed of the single a and a ring of c subunits. Experimental work in this system has been greatly facilitated by the ability to separate the water-soluble F 1 sector (subunits ␣ 3 ␤ 3 ␥␦⑀) from the membrane-embedded F 0 sector (subunits ab 2 c ring ), with retention of ATP hydrolysis function in the former and of proton conduction in the latter. For recent reviews of structure and function of ATP synthase see Refs. 1-4.Based on kinetic measurements, Boyer and co-workers (5, 6) hypothesized that the affinity of catalytic sites for P i is increased by the proton gradient, and currently the concept that P i binding is enhanced by proton gradient-driven subunit rotation appears well supported. Significant P i binding to Escherichia coli F 1 catalytic sites was not measurable by direct binding assay using radioactive P i (7). The K d value for P i binding was Ͼ10 mM as measured by competition with Mg-AMPPNP or ATP in fluorescence titrations using the ␤Y331W mutant enzyme (8 -10). Calculations based on unisite catalysis kinetics showed that K d P i in unisite catalysis is ...

Candida albicans is an opportunistic human fungal pathogen that causes candidiasis. As healthcare has been improved worldwide, the number of immunocompromised patients has been increased to a greater extent and they are highly susceptible to various pathogenic microbes and C. albicans has been prominent among the fungal pathogens. The complete genome sequence of this pathogen is now available and has been extremely useful for the identification of repertoire of genes present in this pathogen. The major challenge is now to assign the functions to these genes of which 13% are specific to C. albicans. Due to its close relationship with yeast Saccharomyces cerevisiae, an edge over other fungal pathogens because most of the technologies can be directly transferred to C. albicans from S. cerevisiae and it is amenable to mutation, gene disruption, and transformation. The last two decades have witnessed enormous amount of research activities on this pathogen that leads to the understanding of host-parasite interaction, infections, and disease propagation. Clearly, C. albicans has emerged as a model organism for studying fungal pathogens along with other two fungi Aspergillus fumigatus and Cryptococcus neoformans. Understanding its complete life style of C. albicans will undoubtedly be useful for developing potential antifungal drugs and tackling Candida infections. This will also shed light on the functioning of other fungal pathogens.

Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F 1 -ATPase by binding at the β-subunit DELSEED motif of F 1 F o 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 F 1 and membrane bound F 1 F o E. 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% to 70%. MRP-amide was also the most potent inhibitor on molar scale (IC 50 ~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 F 1 F o membrane preparations as well as in isolated purified F 1 . Interestingly, growth of Escherichia 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 melittinamide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F 1 -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. KeywordsF 1 F o -ATP synthase; F 1 -ATPase; E. coli ATP synthase; Antimicrobial peptides; Amphibian; Enzyme inhibitors F 1 F o -ATP synthase is the primary source of cellular energy production in animals, plants, and almost all microorganisms by oxidative or photophosphorylation. The ATP synthase enzyme is highly conserved and structurally similar in all organisms. This enzyme is the smallest known biological nanomotor and is composed of two rotary sectors, F 1 and F o . In its simplest form in Escherichia coli ATP synthase contains eight different subunits namely α 3 ß 3 γδεab 2 c 10 with a total molecular mass of ~530 kDa. F 1 corresponds to α 3 ß 3 γδε -and F o to ab 2 c 10 . The reversible processess of ATP hydrolysis and synthesis occur on three catalytic sites in the F 1 sector, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. whereas proton transport occurs through the membrane embedded F o [1][2]. An important feature of the molecular mechanism of ATP synthase is that a "rotor" made up...

This paper describes the role of ␣-subunit VISIT-DG sequence residues ␣Ser-347 and ␣Gly-351 in catalytic sites of Escherichia coli F 1 F o ATP synthase. X-ray structures show the very highly conserved ␣-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues ␣Arg-376, ␤Arg-182, ␤Lys-155, and ␤Arg-246 in the phosphate-binding subdomain. Mutations ␣S347Q and ␣G351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F 1 F o in membranes by 100-and 150-fold, respectively, whereas ␣S347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of ␣S347Q mutant was not inhibited, and the ␣S347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and ␣G351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and ␣G351Q mutant ATPase essentially completely, ATPase in ␣S347A or ␣S347Q mutant was inhibited maximally by ϳ80 -90%, although reaction still occurred at residue ␤Tyr-297, proximal to the ␣-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ␤E (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and ␣G351Q mutant but not in ␣S347Q or ␣S347A mutant. The results demonstrate that ␣Ser-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, ␣Gly-351, although strongly conserved and clearly important for function, appears not to play a direct role.F 1 F o -ATP synthase is the enzyme responsible for ATP synthesis by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. It is the fundamental means of cell energy production in animals, plants, and almost all microorganisms. It works like a nanomotor and is structurally similar in all species. In its simplest form, as in Escherichia coli, it contains eight different subunits distributed in the water-soluble F 1 sector (subunits ␣ 3 ␤ 3 ␥␦⑀) and the membraneassociated F o sector (subunits ab 2 c 10 ). The total molecular size is ϳ530 kDa. In chloroplasts there are two isoforms of subunit b. In mitochondria, there are 7-9 additional subunits, depending on the source, but in toto they contribute only a small fraction of additional mass and may have regulatory roles (1-4).ATP hydrolysis and synthesis occur in the F 1 sector. X-ray structures of bovine enzyme (5) established the presence of three catalytic sites at ␣/␤ subunit interfaces of the ␣ 3 ␤ 3 hexamer. Proton transport occurs through the membrane-embedded F o . The ␥-subunit contains three ␣-helices. Two of these helices form a coiled coil and are located in the central space of the ␣ 3 ␤ 3 hexamer. Proton gradient-driven clockwise rotation of ␥ (as viewed from the membrane) leads to ATP synthesis and anticlockwise rotation ...