Vectorial Proton Transport Research Articles

Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.

The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.

Rates of vectorial proton transport supported by cyclic electron flow during oxygen reduction by illuminated intact chloroplasts

The light-dependent quenching of 9-aminoacridine fluorescence was used to monitor the state of the transthylakoid proton gradient in illuminated intact chloroplasts in the presence or absence of external electron acceptors. The absence of appreciable light-dependent fluorescence quenching under anaerobic conditions indicated inhibition of coupled electron transport in the absence of external electron acceptors. Oxygen relieved this inhibition. However, when DCMU inhibited excessive reduction of the plastoquinone pool in the absence of oxygen, coupled cyclic electron transport supported the formation of a transthylakoid proton gradient even under anaerobiosis. This proton gradient collapsed in the presence of oxygen. Under aerobic conditions, and when KCN inhibited ribulose bisphosphate carboxylase and ascorbate peroxidase, fluorescence quenching indicated the formation of a transthylakoid proton gradient which was larger with oxygen in the Mehler reaction as electron acceptor than with methylviologen at similar rates of linear electron transport. Apparently, cyclic electron transport occured simultaneously with linear electron transport, when oxygen was available as electron acceptor, but not when methylviologen accepted electrons from Photosystem I. The ratio of cyclic to linear electron transport could be increased by low concentrations of DCMU. This shows that even under aerobic conditions cyclic electron transport is limited in isolated intact chloroplasts by excessive reduction of electron carriers. In fact, P700 in the reaction center of Photosystem I remained reduced in illuminated isolated chloroplasts under conditions which resulted in extensive oxidation of P700 in leaves. This shows that regulation of Photosystem II activity is less effective in isolated chloroplasts than in leaves. Assuming that a Q-cycle supports a H(+)/e ratio of 3 during slow linear electron transport, vectorial proton transport coupled to Photosystem I-dependent cyclic electron flow could be calculated. The highest calculated rate of Photosystem I-dependent proton transport, which was not yet light-saturated, was 330 μmol protons (mg chlorophyll h)(-1) in intact chloroplasts. If H(+)/e is not three but two proton transfer is not 330 but 220 μmol (mg Chl H)(-1). Differences in the regulation of cyclic electron transport in isolated chloroplasts and in leaves are discussed.

111 Bioluminescence analysis and numerical evaluation of ATP-synthesis by native and reconstituted membranes containing bacterial ATP-synthase

ATP-synthase is a large membrane protein complex, which plays a key role in the energy metabolism of most organisms. It consists of at least eight types of subunits and can be isolated and purified from several organisms, e.g. bacteria. The enzyme couples two reversible reactions: vectorial proton transport through a membrane and synthesis of the energy rich molecule ATP. Both can be investigated with vesicles from native membranes or with reconstituted liposomes from purified ATPsynthase. The analysis is complicated because ATP-synthase catalyzes ATP-synthesis as well as ATP-hydrolysis. Furthermore the ATP level of membrane samples is influenced by adenylate kinase activities of other enzymes.

On the action spectrum of the photoelectric transients of bacteriorhodopsin in solid-state films

We have measured the time-resolved charge displacement currents of bacteriorhodopsin (BR) from Halobacterium halobium in multilayer arrays of well-oriented purple membranes in photoelectric cells with the sandwich electrode configuration: SnO 2 |BR|Ag. The purple membrane cells were irradiated by dye laser pulses entering the cells through the light-transmitting SnO 2 electrode. The photochemical cycle of BR was found to be reversible in these solid films, and we observed a major decay component for the M intermediate with a decay time of 46 ± 3 ms as measured by the transient optical absorption at 412 nm. We also observed a minor component (≤5% of the total amplitude) with a decay time of 0.97 ± 0.15 s. We measured the early action spectrum for the instrument limited amplitudes of the photoelectric behaviour: this action spectrum differed from the absorption spectrum of the absorbing BR pigment. However, the action spectrum measured 500 μs after flash excitation was in agreement with the absorption spectrum of BR. We attribute the nature of the early action spectrum to the presence of red and blue absorbing species which are not capable of pumping protons across the purple membrane bilayer. We also measured the charge displacement currents when the purple membrane cells were perturbed by small applied voltages with a specific polarity: for conduction of negative charges in the same direction as vectorial proton transport, enhanced transient photocurrents resulted. We interpret these enhanced photocurrents in terms of an enhancement of observed charge separation resulting from a reduced back reaction from the K intermediate of the photocycle.

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.

Preferential interaction parameters for F 1-ATPase from a thermophilic bacterium (PS3)

The proton translocating ATPase of energy transducing membranes couples the vectorial transport of protons through the membrane to synthesize ATP. The enzyme can be divided into an integral membrane portion, F,, and a peripheral portion, called the coupling factor or Fr-ATPase, which contains the catalytic site of ATP synthesis and hydrolysis [l-3]. The Fr portion of the H’-ATPase consists of 5 different subunit species, OL,/~,T,~ and E. The subunits have been isolated of Fr from Escherichia coli, PS3 and CFr [4-61, and their amino acid composition and Mr-values determined [7]. The subunit stoichiometry is still controversial because of incomplete reassembly to a functional Fr of the isolated subunits [7]. Also, the reported J&-values of Fr vary from 350 OOO380 000, e.g., for ECFr [9,10], due to differences in analytical methods and preparations. However, analytical ultracentrifugation data were not treated as a multicomponent system with c2 the mass of the protein (Fr), c3 the added solvent component, e.g., methanol or glycerol, and cr the principal solvent (buffer) with cr in g/ml solution. This paper reports on studies of TFr in 0.01 M Tris-HCI buffer at pH 7.0-8-O in the presence and absence of methanol and glycerol by means of light scattering and differential refractometry experiments, in order to detect any interactions of component three with the protein. Since these organic solvents