Thermodynamics of charge separation of photosystem I in the menA and menB null mutants of Synechocystis sp. PCC 6803 determined by pulsed photoacoustics.

When the biosynthesis of phylloquinone is inhibited in Synechocystis sp. PCC 6803 by interrupting the menA or the menB gene, photosystem I (PS I) recruits plastoquinone-9 (A(P)) to occupy the A(1) sites. In PS I from the menA and menB null mutants, forward electron transfer from the quinone to the FeS clusters occurs approximately 1000 times slower than in wild-type PS I [Semenov, A. Yu., Vassiliev, I. R., van der Est, A., Mamedov, M. D., Zybailov, B., Shen, G., Stehlik, D., Diner, B. A., Chitnis, P. R., and Golbeck, J. H. (2000) J. Biol. Chem. 275, 23429-23438]. To investigate the effect on thermodynamics, the enthalpy and volume changes of charge separation in PS I in the menA and menB mutants were measured using pulsed time-resolved photoacoustics on the nanosecond and microsecond time scales. The observed thermodynamic data are the same for the menA and menB mutants. This is expected because the recruited quinone (A(P)) is the same in both mutants. The volume change of PS I from the mutants following charge separation on both time scales was -17 +/- 2 A(3), less than that of the wild type, -21 A(3). The quantum yield of charge separation was found to be slightly lower (85 +/- 9%) than that of wild-type PS I (96 +/- 10%). The observed reaction is assigned to the formation of P(700)(+)A(P)(-) from P(700)*A(P). An enthalpy change (DeltaH) of -0.69 +/- 0.07 eV was obtained for this reaction. In contrast, a larger enthalpy change -0.8 eV for the formation of P(700)(+)A(1)(-) from P(700)* and an apparent entropy change (TDeltaS, T = 25 degrees C) of -0.2 eV were obtained in wild-type PS I [Hou, H. J. M., and Mauzerall, D. (2006) J. Am. Chem. Soc. 128, 1580-1586]. Taking the free energy to be -0.70 eV in PS I of the mutants, the apparent entropy is close to zero in the mutants. Since the apparent entropy change for the overall reaction of the production of P(700)(+)F(A/B)(-) from P(700)* is very likely the same as that of the wild type, +0.35 eV, this implies that the reaction of P(700)(+)A(P)(-)F(A/B) --> P(700)(+)A(P)F(A/B)(-) in the mutants is almost completely entropy driven (DeltaG = -0.07 eV and TDeltaS = +0.40 eV). These results show that not only the kinetics but also the thermodynamics of electron transfer reactions in PS I are significantly affected by the recruitment of the foreign plastoquinone-9 into the A(1) site.

Association of chlorophyll a/b-binding Pcb proteins with photosystems I and II in Prochlorothrix hollandica.

Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H(2) photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192+/-28 and 139+/-15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb-PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with approximately 14 Pcb light-harvesting proteins, and form the largest known Pcb-PSII supercomplexes.

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration.

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.

Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna.

Energy transfer from light-harvesting carotenoids to chlorophyll is common in photosynthesis, but such antenna pigments have not been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a proton-pumping retinal protein/carotenoid complex in the eubacterium Salinibacter ruber. The wavelength dependence of the rate of pumping and difference absorption spectra measured under a variety of conditions indicate that this protein contains two chromophores, retinal and the carotenoid salinixanthin, in a molar ratio of about 1:1. The two chromophores interact strongly, and light energy absorbed by the carotenoid is transferred to the retinal with a quantum efficiency of approximately 40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill transmembrane proton transport.

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude-Nernst equation.

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1-10 mus time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (-26 A(3)) and reaction enthalpy (-0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, -1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of approximately 4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe(4)S(4))(Cys(4)) with charge change from -1 to -2. The small volume change in PS II is explained by sub-mus electron transfer from Y(Z) anion to P(680) cation, in which charge is only moved from the Y(Z) anion to the Q(A) with no charge separation or with rapid proton transfer from oxidized Y(Z) to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P(680) cation.