Holger Lill

ATP SYNTHASE: AN ELECTROCHEMICAL TRANSDUCER WITH ROTATORY MECHANICS

ATP synthase (F0F1-ATPase) uses proton- or sodium-motive force to produce ATP form ADP and P(i). Three lines of experiment have recently demonstrated large-scale intersubunit rotation during ATP hydrolysis by F1. We discuss how ion flow through the membrane-intrinsic portion, F0, may generate torque and how this might be transmitted between stator and rotor to finally expel spontaneously formed ATP from F1 into water.

Selectivity of TMC207 towards Mycobacterial ATP Synthase Compared with That towards the Eukaryotic Homologue

ABSTRACT The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC50] of >200 μM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC50 of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.

Transport of cytochrome c derivatives by the bacterial Tat protein translocation system

An experimental system developed previously for the heterologous expression of c‐type cytochromes in Escherichia coliQ1has been adapted to monitor protein transfer across the bacterias cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec‐specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat‐mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.

Probing the interaction of the diarylquinoline TMC207 with its target mycobacterial ATP synthase.

Infections with Mycobacterium tuberculosis are substantially increasing on a worldwide scale and new antibiotics are urgently needed to combat concomitantly emerging drug-resistant mycobacterial strains. The diarylquinoline TMC207 is a highly promising drug candidate for treatment of tuberculosis. This compound kills M. tuberculosis by binding to a new target, mycobacterial ATP synthase. In this study we used biochemical assays and binding studies to characterize the interaction between TMC207 and ATP synthase. We show that TMC207 acts independent of the proton motive force and does not compete with protons for a common binding site. The drug is active on mycobacterial ATP synthesis at neutral and acidic pH with no significant change in affinity between pH 5.25 and pH 7.5, indicating that the protonated form of TMC207 is the active drug entity. The interaction of TMC207 with ATP synthase can be explained by a one-site binding mechanism, the drug molecule thus binds to a defined binding site on ATP synthase. TMC207 affinity for its target decreases with increasing ionic strength, suggesting that electrostatic forces play a significant role in drug binding. Our results are consistent with previous docking studies and provide experimental support for a predicted function of TMC207 in mimicking key residues in the proton transfer chain and blocking rotary movement of subunit c during catalysis. Furthermore, the high affinity of TMC207 at low proton motive force and low pH values may in part explain the exceptional ability of this compound to efficiently kill mycobacteria in different microenvironments.

Cross-linking of engineered subunit delta to (alphabeta)3 in chloroplast F-ATPase

Ser → Cys mutations were introduced into subunit δ of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered δ subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the δ subunit (CF1(−δ)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1. Modification of the engineered δ subunit with photolabile cross-linkers, binding of δ to CF1(−δ), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1. By chemical hydrolysis of cross-linked CF1, it was shown that δS10C was cross-linked within the N-terminal 62 residues of subunit β. δS57C, δS82C, and δS166C were cross-linked within the N-terminal 192 residues of subunit α. Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis. Functional reconstitution, however, seemed to be impaired.

ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction

ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.

Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli.

C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. colis maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin.

Proton channel of the chloroplast ATP synthase, CF0: Its time-averaged single-channel conductance as function of pH, temperature, isotopic and ionic medium composition

SummaryThe proton-driven ATP synthase of chloroplasts is composed of two elements, CF0 and CF1. The membrane bound CF0 conducts protons and the peripheral CF1 interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF1 was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CF0. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CF0 as a low-impedance access for protons to the coupling site in CF0CF1. On the other hand, it was too large to be readily reconciled with current concepts of proton supply to and proton conduction through the channel.We studied the time-averaged single-channel conductance of CF0 under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results: (i) CF0 was proton-specific even against a background of 300mm monovalent or 30mm divalent catins. (ii) While the conductance of CF0 was pH/pD-independent in the range from 5.6–8.0, in D2O it was lower by a constant factor of 1.7 than in H2O (iii) Addition of glycerol diminished the conductance and abolished the isotope effect. (iv) The Arrhenius activation energy was 42 kJ/mol and thus intermediate between the ones found for the water-filled pore, gramicidin (30 kJ/mol), and the mobile carrier, valinomycin (65 kJ/mol).The results implied that CF0 is endowed with an extremely proton-specific (107-fold) selectivity filter. Its conductance is very high, and its conduction cycle is not necessarily rate limited by a protolytic reaction. The mechanisms of rapid proton supply to the channel mouth and of proton conduction remained enigmatic.

Biomaterial engineered electrodes for bioelectronics

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase, and, for comparison, gramicidin

SummaryWe previously introduced a flash spectrophotometric method to analyze proton conduction by CF0 in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & W. Junge, 1986,Eur. J. Biochem.160:627–634). The unit conductance of CF0, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channelG, and the second denoting the average number of active channels per vesiclen. This technique was applied to CF0, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable wasy, i.e.n by addition of known inhibitors of F0-type proton channels andG via the temperature. for gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF0, whose conductance so far has not been accessible to other, more conventional approaches. The time-averaged unit conductance of CF0 was about 1 pS, equivalent to the turnover of 6×105 H+/(CF0·sec) at 100 mV driving force.