Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane.

The transmembrane electrical potential (deltaphi), the proton flux (H+), the rate of electron transport (e), the pH gradient (deltapH) and the rate of phosphorylation (ATP) were measured in chloroplasts of spinach. Photosynthesis was excited periodically with flashes of variable frequencies and intensities. A new method is described for determining the rate of electron transport and proton flux. Under conditions where the rate of electron transport and proton flux are not pH controlled the following correlations were found in the range 50 mV less than or equal to deltaphi less than or equal to 125 mV and 1.8 less than or equal to deltapH less than or equal to 2.7: (1) The pH gradient, deltapH, increases with H+ independently of Phout between 7-9. (2) The rate of phosphorylation, ATP, depends exponentially on deltapH (at constant deltaphi) and is independent of pHout between 7-9. (3) The rate of phosphorylation, ATP, depends also on deltaphi (at constant deltapH and at constant proton flux H+). (4) The proton flux via the ATPase pathway, Hp+, depends non-linearly on the ratio of the proton concentrations: Hp+ approximately (Hin+/Hout+)b, (b=2.3--2.6). The proton flux via the basal pathway, Hb+, depends linearly on the ratio of the proton concentrations: Hb+ approximately (Hin/Hout). (5) The ratio deltaH+/ATP (e/ATP, i.e. the ratio of the total proton flux, Hp+ + Hb+, and the rate of ATP formation, ATP, depends strongly on deltaphi and on deltapH. The ratio is deltaH+/ATP approximately 3 (e/ATP approximately 1.5) at deltapH 2.7 and deltaphi = 125 mV. (6) It is supposed that the reason for the dependence of deltaH+/ATP on deltaphi anddeltapH is the different functional dependence of the basal proton flux Hb+ and the phosphorylating proton flux Hp+ on deltapH and deltaphi. The calculation of deltaH+/ATP on the basis of this assumption is in fair agreement with the experimental values. Also the "threshold" effects can be explained in this way. (7) The ratio of deltaHp+/ATP, i.e. the ratio of the phosphorylating proton flux Hp+ and ATP, is deltaHp+/ATP APPROXIMATELY 2.4.

On the extent of the electrical potential across the thylakoid membrane induced by continuous light in Chlorella cells.

The extent of the electrical potential delta phi(SS) across the thylakoid membrane of Chlorella cells was estimated under steady state conditions. This has been achieved by comparing the absorption change which occurs after continuous light is switched off with a calibrated field indicating absorption change induced by flash light. Under saturating light conditions delta phi(SS) is in the order of 100 mV.

Relation between the initial kinetics of ATP synthesis and of conformational changes in the chloroplast ATPase studied by external field pulses.

ATP formation and the energy-dependent release of tightly bound [14C]-adenine nucleotides from the chloroplast coupling factor CF1 has been studied as a function of the time of energization of the membrane in the range of 500 mus up to 60 ms. The high time resolution was achieved because the energization was generated artificially by external electric field pulses. Applying external electric field pulses to a chloroplast suspension induces an electric potential difference across the thylakoid membrane. The following results were obtained: (1) The amount of ATP generated increases linearly with the time of energization. The steady-state rate of ATP formation is reached in less than 500 mus. (2) A fraction of the adenine nucleotides tightly bound to CF1 is released on energization with a half-rise-time of about 2 ms. The size of the fraction, i.e., the amplitude of the fast phase of the release, increases with the magnitude of the induced transmembrane electric potential difference. A further slow release is superimposed. (3) The initial rate of the release of adenine nucleotides is practically identical with the rate of ATP formation. It is concluded that the release of tightly bound nucleotides monitors an initial conformational change by which the ATPase turns from an inactive into an activated state. For the explanation of the results a reaction scheme is proposed which takes into account a preceding activation of the ATPase.

Electrical potential changes, H+ translocation and phosphorylation induced by short flash excitation in Rhodopseudomonas sphaeroides chromatophores.

1. The basal decay of the carotenoid shift of chromatophores from photosynthetic bacteria following short flash excitation is approximately biphasic. The decay indicates the dissipation of the transmembrane electrical potential. 2. The H+ efflux following rapid H+ binding after a flash, measured from the colour change of added cresol red, shows very similar kinetics to the carotenoid shift decay suggesting that the dissipation of the electric potential decay is a consequence of the H+ efflux. 3. The electric potential decay is stimulated when the chromatophore suspension is supplemented with ADP and Pi (in either the presence or absence of antimycin A). 4. The stimulated electric potential decay by ADP and Pi has a similar pH dependence to that of phosphorylation in continuous light. 5. The stimulation of the electric potential decay by ADP and Pi is reversed, by aurovertin, an antibiotic which inhibits phosphorylation. 6. The stimulation of the electric potential decay by ADP+Pi is also reversed by the inhibitors oligomycin and venturicidin. These inhibitors, but not aurovertin, also inhibit the fast phase of the decay under non-phosphorylating conditions. 7. Valinomycin accelerates the overall rate of decay of the electric potential, inhibits the ADP and Pi stimulated electric potential decay, and inhibits the flash-induced phosphorylation. The decay rate of the H+ efflux however, is slower in the presence of this ionophore. 8. Nigericin-type ionophores accelerate the overall decay rate of the H+ efflux and inhibit the ADP and Pi stimulated electric potential decay. The basal rate of the electric potential decay is unaffected by treatment with these ionophores. 9. When a coupling factor associated with the chromatophore ATPase is removed from the membrane, both the stimulation of the electric potential decay by ADP and Pi and ADP phosphorylation are inhibtied. Both reactions are completely restored after reconstitution with the crude coupling factor extract. The basal electric potential decay rate is not affected by the removal of coupling factor.

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+/ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation.

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and Pi of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H+ consumption during the synthesis of ATP. The ratio of H+ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 H+/ATP. 2. The stimulation of the electrical potential decay by ADP and Pi was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No 'critical' or 'threshold' potential was observed. 3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle. 4. The stimulation of the electrical potential decay by ADP and Pi is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H+-release on the inside of the chromatophore vesicles following a flash.

First photosystem II crystals capable of water oxidation.

Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.

The plastoquinone pool as possible hydrogen pump in photosynthesis.

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e-) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions: (1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation. These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e-) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.

Effect of electric fields on the absorption spectrum of dye molecules in lipid layers. V. Refined analysis of the field-indicating absorption changes in photosynthetic membranes by comparison with electrochromic measurements in vitro.

The comparison of light-induced absorption changes in photosynthesis with electrochromic spectra of the isolated pigments in vitro is renewed more thoroughly and described in detail, involving new measurements of the linear electrochromism of oriented chlorophyll beta [6]. 1. The coincidence of the maxima and minima in the in vivo spectrum with those in the in vitro superposition is better than in previous studies [4]. 2. The molar ratio of the pigments now used for the superposition of the in vitro spectra is the same as that in vivo. 3. From this and from surface-pressure/area diagrams of the chlorophylls on a water surface, conclusions are drawn concerning the preferential orientations of the dipole moment differences of the red and blue absorption bands of the bulk chlorophylls in the membrane. 4. From the comparison of the electrochromism of the carotenoids with the absorption change at 520 nm in vivo, it is concluded that the bulk of the carotenoids are oriented at a rather flat angle in the membrane (approximately 16 degrees).

The functional unit of electrical events and phosphorylation in chromatophores from Rhodopseudomonas sphaeroides.

1. From electron micrographs of chromatophores from Rhodopseudomonas spaeroides and from the estimated bacteriochlorophyll per chromatophore was estimated. The mean diameter of the chromatophore vesicles was 600 A. 2. The decay of the flash-induced electric potential across the chromatophore membrane measured by the carotenoid band shift was 20% accelerated by about one valinomycin molecule per 4700 bacteriochlorophyll, i.e. by one ionophore molecule per chromatophore. 3. The inhibition of the flash-induced ATP formation by valinomycin followed a similar pattern to the accelerated decay of the electric potential. 4. The single turnover flash yield of ATP synthesis gave a mean value of one ATP per 1470 bacteriochlorophyll or about 3 ATP per vesicle. 5. With regard to the partitioning of the ionophore between the membrane (85%) and aqueous phase (15%) we conclude that one molecule of valinomycin per chromatophore is sufficient to begin to collapse the electrical potential and inhibit ATP synthesis. It is therefore suggested that the membrane potential is an essential component of the energized state which is used for phosphorylation. The results correspond to those obtained for the 100-fold larger vesicles in chloroplasts (thylakoids) where one molecule of ioophore is also sufficient to quench both events.

Photosystem I of Synechococcus elongatus at 4 A resolution: comprehensive structure analysis.

An improved structural model of the photosystem I complex from the thermophilic cyanobacterium Synechococcus elongatus is described at 4 A resolution. This represents the most complete model of a photosystem presently available, uniting both a photosynthetic reaction centre domain and a core antenna system. Most constituent elements of the electron transfer system have been located and their relative centre-to-centre distances determined at an accuracy of approximately 1 A. These include three pseudosymmetric pairs of Chla and three iron-sulphur centres, FX, FA and FB. The first pair, a Chla dimer, has been assigned to the primary electron donor P700. One or both Chla of the second pair, eC2 and eC'2, presumably functionally link P700 to the corresponding Chla of the third pair, eC3 and eC'3, which is assumed to constitute the spectroscopically-identified primary electron acceptor(s), A0, of PSI. A likely location of the subsequent phylloquinone electron acceptor, QK, in relation to the properties of the spectroscopically identified electron acceptor A1 is discussed. The positions of a total of 89 Chla, 83 of which constitute the core antenna system, are presented. The maximal centre-to-centre distance between antenna Chla is < or = 16 A; 81 Chla are grouped into four clusters comprising 21, 23, 17 and 20 Chla, respectively. Two "connecting" Chla are positioned to structurally (and possibly functionally) link the Chla of the core antenna to those of the electron transfer system. Thus the second and third Chla pairs of the electron transfer system may have a dual function both in energy transfer and electron transport. A total of 34 transmembrane and nine surface alpha-helices have been identified and assigned to the 11 subunits of the PSI complex. The connectivity of the nine C-terminal (seven transmembrane, two "surface") alpha-helices of each of the large core subunits PsaA and PsaB is described. The assignment of the amino acid sequence to the transmembrane alpha-helices is proposed and likely residues involved in co-ordinating the Chla of the electron transfer system discussed.

A common ancestor for oxygenic and anoxygenic photosynthetic systems: a comparison based on the structural model of photosystem I.

The 4 A structural model of photosystem I (PSI) has elucidated essential features of this protein complex. Inter alia, it demonstrates that the core proteins of PSI, PsaA and PsaB each consist of an N-terminal antenna-binding domain, and a C-terminal reaction center (RC)-domain. A comparison of the RC-domain of PSI and the photosynthetic RC of purple bacteria (PbRC), reveals significantly analogous structures. This provides the structural support for the hypothesis that the two RC-types (I and II) share a common evolutionary origin. Apart from a similar set of constituent cofactors of the electron transfer system, the analogous features include a comparable cofactor arrangement and a corresponding secondary structure motif of the RC-cores. Despite these analogies, significant differences are evident, particularly as regards the distances between and the orientation of individual cofactors, and the length and orientation of alpha-helices. Inferred roles of conserved amino acids are discussed for PSI, photosystem II (PSII), photosystem C (PSC, green sulfur bacteria) and photosystem H (PSH, heliobacteria). Significant sequence homology between the N-terminal, antenna-binding domains of the core proteins of type-I RCs, PsaA, PsaB, PscA and PshA (of PSI, PSC and PSH respectively) with the antenna-binding subunits CP43 and CP47 of PSII indicate that PSII has a modular structure comparable to that of PSI.

Functional mechanism of water splitting photosynthesis.

A personal account is given on physico-chemical aspects of photosynthesis. The article starts with the way I entered the field of photosynthesis. Then, selected results from our research group are discussed. Three methods used for functional analysis in our laboratory are described: the repetitive flash spectroscopy; the electrochromic volt- and ammeter; and the membrane energization by a battery. Our subsequent studies deal with the two photoreaction centers, the primary charge separation, the plastoquinones as a transmembrane link between the two centers and the vectorial electron- and proton pathways. The results led to a picture of the elementary functional mechanism of the molecular machinery in the thylakoid membrane. The perspective then focuses on the coupling between the electric field, protons and phosphorylation. This section is followed by our observations and analysis of the mechanism of water cleavage and its coupling with the functioning of reaction center II. Finally, information is provided on structural aspects of the two reaction centers. The article ends with a retrospect.