Summary. We previously introduced a flash spectrophotometricmethod to analyze proton conduction by CF0 in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Sch6n-knecht & 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 channel G, and the second denoting the average number of active channels per vesicle h-. 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 ways, i.e. g by addition of known inhibitors of F0-type proton channels and G 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 timeaveraged unit conductance of CF0 was about 1 pS, equivalent to the turnover of 6 x l0 t H+/(CF0 -sec) at 100 mV driving force.

The size of the function unit of electrical events in thylakoid membranes was estimated by the minimum amount of gramicidin needed to discharge the flash light generated electrical potential difference. Early flash spectroscopic measurements have indicated that a single gramicidin dimer operates on an electrical function unit containing at least 2 × 105 chlorophyll molecules [1]. In this study we present gramicidin titrations with more intact thylakoid preparations which revealed a more than hundred‐fold greater lower limit for the electric unit size, namely 5 × 107 chlorophyll molecules. It is conceivable that the whole complicated thylakoid structure inside a chloroplast constitutes a single electric unit. It comprises more than 2 × 108 chlorophyll molecules in an area of more than 400 μm2.

The proton-driven ATP synthase of chloroplasts is composed of two elements, CFo and CFt. The membrane bound CFo conducts protons and the peripheral CFt interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF~ was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CFo. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CFo as a low-impedance access for protons to the coupling site in CFoCF1. 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 CFo under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results:(i) CFo was proton-specific even against a background of 300 mM monovalent or 30 mM divalent cations. (ii) While the conductance of CFo was pH/pD-independent in the range from 5.6-8.0, in D20 it was lower by a constant factor of 1.7 than in H20. (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 CFo 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.

The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5 x 10(14) cm2/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the up-concentration of gramicidin in the small fractional area of protein-free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nM (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mM NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.

The chloroplast H+-ATPase (CF,CF,) was isolated and reconstituted Into hpld vesicles by dlalysls tecinnque Vesicles were fused by dehydration/ rehydratlon to obtain cell-size Itposomes, which were studied by patch~lamp techniques Smgle-channel actzvity was observed with several conductance levels m the range of some 10 pS (100 mM KCI) In contrast to Intact CF,, which conducts protons, only (even at pH S), these channels were permeable for potassmm and sodmm Venturicidm, which blocks proton flow through intact CF,,, here greatly decreased the smgle-channel open probabdlty Subumt III of CF,,, alone, yielded cation channels resemblmg the former Our tentative mterpretatlon IS, that dearrangement or fragmentation of CF, caused the potassmm and sodmm permeablhty, which, however, 1s suppressed m intact CF,