Thermodynamics of primary photosynthesis.

The thermodynamics of photosynthesis has been much discussed, but recent articles have pointed to some confusion on the subject. The aim of this review is to clarify a limited part of this state of affairs.

Efficiency of photosynthesis in a Chl d-utilizing cyanobacterium is comparable to or higher than that in Chl a-utilizing oxygenic species.

The cyanobacterium Acaryochloris marina uses chlorophyll d to carry out oxygenic photosynthesis in environments depleted in visible and enhanced in lower-energy, far-red light. However, the extent to which low photon energies limit the efficiency of oxygenic photochemistry in A. marina is not known. Here, we report the first direct measurements of the energy-storage efficiency of the photosynthetic light reactions in A. marina whole cells, and find it is comparable to or higher than that in typical, chlorophyll a-utilizing oxygenic species. This finding indicates that oxygenic photosynthesis is not fundamentally limited at the photon energies employed by A. marina, and therefore is potentially viable in even longer-wavelength light environments.

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: a pulsed photoacoustic study of electron transfer in photosystem I reveals a similarity to bacterial reaction centers in both volume change and entropy.

The thermodynamic properties of electron transfer in biological systems are far less known in comparison with that of their kinetics. In this paper the enthalpy and entropy of electron transfer in the purified photosystem I trimer complexes from Synechocystis sp. PCC 6803 have been studied, using pulsed time-resolved photoacoustics on the 1 micros time scale. The volume contraction of reaction centers of photosystem I, which results directly from the light-induced charge separation forming P(700+F(A)/F(B-) from the excited-state P700*, is determined to be -26 +/- 2 A3. The enthalpy of the above electron-transfer reaction is found to be -0.39 +/- 0.1 eV. Photoacoustic estimation of the quantum yield of photochemistry in the purified photosystem I trimer complex showed it to be close to unity. Taking the free energy of the above reaction as the difference of their redox potentials in situ allows us to calculate an apparent entropy change (TDeltaS) of +0.35 +/- 0.1 eV. These values of DeltaV and TDeltaS are similar to those of bacterial reaction centers. The unexpected sign of entropy of electron transfer is tentatively assigned, as in the bacterial case, to the escape of counterions from the surface of the particles. The apparent entropy change of electron transfer in biological system is significant and cannot be neglected.

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in manganese-depleted photosystem II core complexes.

We have previously reported the thermodynamic data of electron transfer in photosystem I using pulsed time-resolved photoacoustics [Hou et al. (2001) Biochemistry 40, 7109-7116]. In the present work, using preparations of purified manganese-depleted photosystem II (PS II) core complexes from Synechocystis sp. PCC 6803, we have measured the DeltaV, DeltaH, and estimated TDeltaS of electron transfer on the time scale of 1 micros. At pH 6.0, the volume contraction of PS II was determined to be -9 +/- 1 A3. The thermal efficiency was found to be 52 +/- 5%, which corresponds to an enthalpy change of -0.9 +/- 0.1 eV for the formation of the state P680+Q(A-) from P680*. An unexpected volume expansion on pulse saturation of PS II was observed, which is reversible in the dark. At pH 9.0, the volume contraction, the thermal efficiency, and the enthalpy change were -3.4 +/- 0.5 A3, 37 +/- 7%, and -1.15 +/- 0.13 eV, respectively. The DeltaV of PS II, smaller than that of PS I and bacterial centers, is assigned to electrostriction and analyzed using the Drude-Nernst equation. To explain the small DeltaV for the formation of P680+Q(A-) or Y(Z*)Q(A-), we propose that fast proton transfer into a polar region is involved in this reaction. Taking the free energy of charge separation of PS II as the difference between the energy of the excited-state P680* and the difference in the redox potentials of the donor and acceptor, the apparent entropy change (TDeltaS) for charge separation of PS II is calculated to be negative, -0.1 +/- 0.1 eV at pH 6.0 (P680+Q(A-)) and -0.2 +/- 0.15 eV at pH 9.0 (Y(Z*)Q(A-)). The thermodynamic properties of electron transfer in PS II core reaction centers thus differ considerably from those of bacterial and PS I reaction centers, which have DeltaV of approximately -27 A3, DeltaH of approximately -0.4 eV, and TDeltaS of approximately +0.4 eV.

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in the intact cells of the cyanobacterium Synechocystis PCC 6803.

The volume and enthalpy changes for charge transfer in the 0.1-10 micros time window in photosynthetic reaction centers of the intact cells of Synechocystis PCC 6803 were determined using pulsed, time-resolved photoacoustics. This required invention of a method to correct for the cell artifact at the temperature of maximum density of water caused by the heterogeneous system. Cells grown under either white or red light had different PS I/PS II molar ratios, approximately 3 and approximately 1.7, respectively, but invariable action spectra and effective antenna sizes of the photosystems. In both cultures, the photoacoustic measurements revealed that their thermodynamic parameters differed strongly in the spectral regions of predominant excitation of PS I (680 nm) and PS II (625 nm). On correcting for contribution of the two photosystems at these wavelengths, the volume change was determined to be -27 +/- 3 and -2 +/- 3 A3 for PS I and PS II, respectively. The energy storage on the approximately 1 micros time scale was estimated to be 80 +/- 15% and 45 +/- 10% per trap in PS I and PS II, respectively. These correspond to enthalpies of -0.33 +/- 0.2 and -1 +/- 0.2 eV for the assumed formation of ion radical pairs P700+F(AB-) and Y(Z*)P680Q(A-), respectively. Taking the free energy of the above reactions as the differences of their redox potentials in situ, apparent entropy changes were estimated to be +0.4 +/- 0.2 and -0.2 +/- 0.2 eV for PS I and PS II, respectively. These values are similar to that obtained in vitro for the purified reaction center complexes on the microsecond time scale [Hou et al. (2001) Biochemistry 40, 7109-7116, 7117-7125]. The constancy of these thermodynamic values over a 2-fold change of the ratio of PS I/PS II is support for this method of in vivo analysis. Our pulsed PA method can correct the "cell" or heterogeneous artifact and thus opens a new route for studying the thermodynamics of electron transfer in vivo.

A simple light-driven transmembrane proton pump.

Light-induced lipophilic porphyrin/aqueous acceptor charge separation across a single lipid-water interface can pump protons across the lipid bilayer when the hydrophobic weak acids, carbonylcyanide m-chlorophenylhydrazone and its p-trifluoromethoxyphenyl analogue, are present. These compounds act as proton carriers across lipid bilayers. In their symmetric presence across the bilayer, the positive currents and voltages produced by the photogeneration of porphyrin cations are replaced by larger negative currents and voltages. The maximum negative current and voltage occur at the pH of maximum dark conductance. The reversed larger current and voltage show a positive ionic charge transport in the same direction as the electron transfer. This transport can form an ion concentration gradient. The movement of protons is verified by an unusual D2O isotope effect that increases the negative ionic current by 2- to 3-fold. These effects suggest that an interfacial pK shift of the weak acid caused by the local electric field of photoformed porphyrin cations/acceptor anions functions as the driving force. The estimated pumping efficiency is 10-30%. Time-resolved results show that proton pumping across the bilayer occurs on the millisecond time scale, similar to that of biological pumps. This light-driven proteinless pump offers a simple model for a prebiological energy transducer.

Evidence for ion chain mechanism of the nonlinear charge transport of hydrophobic ions across lipid bilayers.

The conductivity across a lipid bilayer by tetraphenylborate anion is increased 10-fold on the photoformation of lipophilic porphyrin cations. The cations alone have negligible conductivity. This nonlinear photogenerated increase of ion conductivity is termed the photogating effect. Substitution of H by Cl in the para position of tetraphenylborate leads to a 100-fold enhancement of conductivity, whereas the dark conductivities for this and other substituted borates are the same. Moreover, the halo-substituted borates show a large enhancement of conductivity in the low concentration range (10(-8) M), whereas that of tetraphenylborate is small and space charge is negligible. The enhanced ion conductivity has great structural sensitivity to the structure of the anion, the cation, and the lipid, whereas the partition coefficient of all the borates and the concentration of photoformed cations are only slightly affected. The photogated ion transport has a twofold larger activation energy than transport in the dark. Time-resolved photocurrents and voltages demonstrate that the translocation rate of the porphyrin cation is also enhanced 100-fold by the Cl-borate anion but only 10-fold by the H-borate anion. For these reasons the nonlinear gating effect cannot be explained by electrostatics alone, but requires an ion chain or ion aggregate mechanism. Kinetic modeling of the photoinduced current with a mixed cation-anion ion chain can fit the data well. The photogating effect allows the direct study of ion interactions within the bilayer.

Charge transfer across a single lipid-water interface causes ion pumping across the bilayer.

The photoformation of magnesium-porphyrin cations (P+) at a single lipid bilayer-water interface can pump lipophilic borate anions completely across the lipid bilayer and causes an actual reversal of the photovoltage. The system consists of a lipid bilayer containing magnesium octaethylporphyrin, an aqueous or interfacial electron acceptor on one side, and chloro- or fluoro-substituted tetraphenylborate in both aqueous electrolyte solutions. With 1-micros pulsed illumination, an immediate positive photovoltage is observed, which decreases on the microsecond and millisecond time scales. On the time scale of seconds, as the P+ cation concentration decays in reverse electron transfer, the voltage swings negative to a value almost equal to its initial value and finally decays with a half-time (approximately 20 s) longer than the time constant of the system (approximately 5 s). Thus, an ion gradient across the membrane is formed, trapped by the nonlinear relation between ion mobility and ion concentration. Continuous light illumination confirms that negative charge moves in the direction opposite that of the initial photoinduced electron transfer. Steady-state measurements indicate an ion pumping efficiency of approximately 30%. This simple mechanism may be a progenitor of photobiological ion pumps.

Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics.

We have studied the photoinduced volume changes, energetics, and kinetics in the early steps of the bacteriorhodopsin (BR) photocycle with pulsed, time-resolved photoacoustics. Our data show that there are two volume changes. The fast volume change ( < or = 200 ns) is an expansion (2.5 +/- 0.3 A3/molecule) and is observed exclusively in the purple membrane (PM), vanishing in the 3-[(3-cholamidopropyl)-dimethylammonio] -1-propane-sulfonate-sulfonate-solubilized BR sample; the slow change (approximately 1 micros) is a volume contraction (-3.7 +/- 0.3 A3/molecule). The fast expansion is assigned to the restructuring of the aggregated BR in the PM, and the 1-micros contraction to the change in hydrogen bonding of water at Asp 212 (Kandori et al. 1995. J. Am. Chem. Soc. 117:2118-2119). The formation of the K intermediate releases most of the absorbed energy as heat, with delta Hk = -36 +/- 8 kJ/mol. The activation energy of the K --> L step is 49 +/- 6 kJ/mol, but the enthalpy change is small, -4 +/- 10 kJ/mol. On the time scale we studied, the primary photochemical kinetics, enthalpy, and volume changes are not affected by substituting the solvent D2O for H2O. Comparing data on monomeric and aggregated BR, we conclude that the functional unit for the photocycle is the BR monomer, because both the kinetics (rate constant and activation energy) and the enthalpy changes are independent of its aggregation state.

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae.

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.

Bill Arnold's concept of solid state photosynthesis and his discoveries.

Bill Arnold's concept of photosynthesis as a solid state, as opposed to solution, phenomenon led him to an amazing series of fundamental discoveries.

Effects of Iron Limitation on Photosystem II Composition and Light Utilization in Dunaliella tertiolecta.

The effects of iron limitation on photosystem II (PSII) composition and photochemical energy conversion efficiency were studied in the unicellular chlorophyte alga Dunaliella tertiolecta. The quantum yield of photochemistry in PSII, inferred from changes in variable fluorescence normalized to the maximum fluorescence yield, was markedly lower in iron-limited cells and increased 3-fold within 20 h following the addition of iron. The decrease in the quantum yield of photochemistry was correlated with increased fluorescence emission from the antenna. In iron-limited cells, flash intensity saturation profiles of variable fluorescence closely followed a cumulative one-hit Poisson model, suggesting that PSII reaction centers are energetically isolated, whereas in iron-replete cells, the slope of the profile was steeper and the calculated probability of energy transfer between reaction centers increased to >0.6. Immunoassays revealed that in iron-limited cells the reaction center proteins, D1, CP43, and CP47, were markedly reduced relative to the peripheral light-harvesting Chl-protein complex of PSII, whereas the [alpha] subunit of cytochrome b559 was about 10-fold higher. Spectroscopic analysis established that the cytochrome b559 peptide did not contain an associated functional heme. We conclude that the photochemical conversion of absorbed excitation energy in iron-limited cells is limited by the number of photochemical traps per unit antenna.

Volume contraction on photoexcitation of the reaction center from Rhodobacter sphaeroides R-26: internal probe of dielectrics.

Reaction centers of Rhodobacter sphaeroides undergo a approximately 20 A3/mole volume contraction in < 50 ns after excitation. The rapid volume change is tentatively assigned to electrostriction. From its magnitude, we infer that the effective dielectric coefficient is 10-15 if the compressibility of the reaction center is similar to that of globular proteins. The volume contraction is not sensitive to replacement of the natural ubiquinone at the QA site by other quinones or to the occupancy of the QB site. The quenching caused by pressure on the reaction centers most likely occurs on a faster time scale than that of electron transfer.

Synthesis of amphipathic porphyrins and their photoinduced electron transfer reactions at the lipid bilayer-water interface.

A one flask synthesis of cis-substituted amphipathic porphyrins is reported. These porphyrins were used to study electrostatic effects on photoinduced electron transfer across the lipid bilayer-water interface. A neutral porphyrin undergoes only dynamic interfacial electron transfer reactions irrespective of charge of the acceptor, although ionic strength effects indicate a negative charge on the porphyrin donor species. A dianionic porphyrin forms an interfacial static complex with a dicationic electron acceptor, methyl viologen, at low ionic strength. The electron transfer rate within the complex is slow, 10(5) approximately 10(6) s-1, which is attributed to a near orthogonal orientation between the donor and the acceptor pi orbitals.

Photoinduced electron transport across a lipid bilayer mediated by C70.

Electron transport across a membrane is central to photosynthesis, to mitochondrial respiration and to the design of molecular systems for solar energy conversion. Relatively few synthetic molecules, however, have been shown to facilitate transport of electrons across a lipid bilayer. We report here that C70 can act as both a photosensitizer for electron transfer from a donor molecule and a mediator for electron transport across a lipid bilayer membrane. The steady-state photocurrent density obtained from the C70-bilayer system is about 40 times higher, at comparable light intensities, than that of the carotene-porphyrinquinone system, previously the most efficient artificial system. The C70-bilayer system has a quantum yield of about 0.04, while the stability (tens of minutes) and turnover number (electrons transported per C70 before decay) of 10(3) are one to three orders of magnitude greater than those of other systems. We anticipate that other higher fullerenes may also provide the basis for efficient transmembrane electron-transport systems.

Photogating of ionic currents across lipid bilayers. Electrostatics of ions and dipoles inside the membrane.

The conductances of the lipophilic ions tetraphenylboride and tetraphenylphosphonium across a lipid bilayer can be increased or decreased, i.e., gated, by the photoformation of closed-shell metalloporphyrin cations within the bilayer. The gating can be effected by pulsed or continuous light or by chemical oxidants. At high concentrations of lipophilic anions where the dark conductance is saturated due to space charge in the bilayer, the photogated conductance can increase 15-fold. The formation of porphyrin cations allows the conductance to increase to its nonspace charge limited value. Conversely, the decrease of conductance in the light of phosphonium cations diminishes toward zero as the dark conductance becomes space charge limited. We present electrostatic models of the space charge limited conductance that accurately fit the data. One model includes an exponentially varying dielectric constant for the polar regions of the bilayer that allows an analytical solution to the electrostatic problem. The exponential variation of the dielectric constant effectively screens the potential and implies that the inside and outside of real dielectric interfaces can be electrically isolated from one another. The charge density, the distance into the membrane of the ions, about one-quarter of its thickness, and the dielectric constant at that position are determined by these models. These calculations indicate that there is insufficient porphyrin charge density to cancel the boride ion space charge and the following article proposes a novel ion chain mechanism to explain these effects. These models indicate that the positive potential arising from oriented carbonyl ester groups, previously used to explain the 10(3)-fold larger conductance of hydrophobic anions over cations, is smaller than previously estimated. However, the synergistic movement of the positive choline group into the membrane can account for the large positive potential.

Photogating of ionic currents across lipid bilayers. Hydrophobic ion conductance by an ion chain mechanism.

The photogating of hydrophobic ion currents across the lipid bilayer membrane allows the direct study of their kinetics by symmetrically forming charge within the membrane and across each interface, rather than across the membrane. We find that the photoinduced conductance continues to increase beyond the region where the tetraphenylboride charge density in the membrane exceeds the estimated porphyrin cation density. This photoconductance is proportional to the tetraphenylboride charge density raised to the second to third power. The risetime of the photogating effect increases with increasing concentration of tetraphenyl boride. The porphyrin cation mobility is increased when the tetraphenylboride anion is present, and low concentrations of tetraphenylphosphonium cation increase the dark conductivity while inhibiting the photoconductivity. The activation energy for both the porphyrin and phosphonium cation induced conductance is more positive than that of the tetraphenylboride conductance. From these results we conclude that in addition to some cancellation of space charge within the membrane, the mechanism of increased conductance involves the transport of these hydrophobic anions via an alternating anion-cation chain, analogous to the Grotthuss mechanism for excess proton conduction in water. This ion chain conductance can be viewed as an evolutionary prototype of an ion channel across the membrane. It also underscores the importance of the counter ion in the transport of large ions such as peptides across the lipid bilayer.

Energy storage of linear and cyclic electron flows in photosynthesis.

The energy storage of photosynthesis in the green alga Chlorella vulgaris was determined by pulsed, time-resolved photoacoustics. The energy storage of the linear electron transfer process in photosynthesis, of cyclic photosystem (PS) I, and possibly of PSII was determined by selection of excitation wavelength and of flash interval. At 695 nm excitation, a rather large cyclic PSI energy storage of 0.68 +/- 0.04 eV/quantum of energy at 8 ms after a 1-mus flash was obtained. This energy remained the same at flash intervals of 0.35 to 60 s and was independent of the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We tentatively assign this energy to the ferredoxin-NADP-reductase-ferredoxin and oxidized cytochrome b(6)/f complexes. An efficient distribution of energy between cyclic and linear systems is obtained with the simple assumption that the turnover time of the cyclic system is slower than that of the linear system. The energy storage of linear electron flow was determined by 655 nm excitation of Chlorella with a short flash interval of 0.35 s per flash. It was calculated to be 0.50 +/- 0.03 eV/hv, close to that expected for oxygen and NADPH formation. The energy storage of PSII is determined by excitation of Chlorella at 655 nm with a long flash interval of 60 s per flash. It was calculated to be 1.07 +/- 0.05 eV/hv, consistent with the energy storage being in S-states and the secondary electron acceptor of PSII with a calculated redox energy of 1.03 eV/hv. In the presence of 1 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the calculated energy storage in PSII is still significant, 0.53 +/- 0.04 eV/hv. This probably indicates a significant cyclic electron flow around PSII. These cyclic flows may contribute considerably to energy storage in photosynthesis.