Photosystem I, when excited in the chlorophyll Qy absorption band, feeds on negative entropy.

It is often suggested that Life may lay outside the normal laws of Physics and particularly of Thermodynamics, though this point of view is refuted by many. As the Living State may be thought of as an open system, often far from equilibrium, most attempts at placing Life under the umbrella of the laws of Physics have been based, particularly in recent years, on non-equilibrium Thermodynamics and particularly the Maximum Entropy Production Principle. In this view it is the dissipation of entropy (heat) which permits the ever increasing complexity of Living Systems in biological evolution and the maintenance of this complexity. However, these studies usually consider such biological entities as whole cells, organs, whole organisms and even Life itself at the entire terrestrial level. This requires making assumptions concerning the Living State, which are often not soundly based on observation and lack a defined model structure. The present study is based on an entirely different approach, in which a classical thermodynamic analysis of a well-defined biological nanoparticle, plant Photosystem I, is performed. This photosynthetic structure, which absorbs light and performs primary and secondary charge separation, operates with a quantum efficiency close to one. It is demonstrated that when monochromatic light is absorbed by the lowest lying electronic transition, the chlorophyll Qy transition, entropy production in the system bath plus entropy changes internal to the system are numerically less than the entropy decrease of the light field. A Second Law violation is therefore suggested for these experimental conditions. This conclusion, while at first sight is supportive of the famous and much discussed statement of Schroedinger, that "Life feeds on negentropy", is analysed and the conditions in which this statement may be considered valid for a Plant Photosystem are defined and delimited. The remarkably high quantum efficiency, leading to minimal entropy production (energy wastage), seems to suggest that evolution of Photosystem I has gone down the road of maximal energy efficiency as distinct from maximal entropy production. Photosystem I cannot be considered a maximum entropy dissipation structure.

Antenna entropy in plant photosystems does not reduce the free energy for primary charge separation.

We have investigated the concept of the so-called "antenna entropy" of higher plant photosystems. Several interesting points emerge: 1. In the case of a photosystemwhich harbours an excited state, the "antenna entropy" is equivalent to the configurational (mixing) entropy of a thermodynamic canonical ensemble. The energy associated with this parameter has been calculated for a hypothetical isoenergetic photosystem, photosystem I and photosystem II, and comes out in the range of 3.5 - 8% of the photon energy considering 680 nm. 2. The "antenna entropy" seems to be a rather unique thermodynamic phenomenon, in as much as it does not modify the free energy available for primary photochemistry, as has been previously suggested. 3. It is underlined that this configurational (mixing) entropy, unlike heat dispersal in a thermal system, does not involve energy dilution. This points out an important difference between thermal and electronic energy dispersal.

A comparison between plant photosystem I and photosystem II architecture and functioning.

Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electron transport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.

Ergodicity, configurational entropy and free energy in pigment solutions and plant photosystems: influence of excited state lifetime.

We examine ergodicity and configurational entropy for a dilute pigment solution and for a suspension of plant photosystem particles in which both ground and excited state pigments are present. It is concluded that the pigment solution, due to the extreme brevity of the excited state lifetime, is non-ergodic and the configurational entropy approaches zero. Conversely, due to the rapid energy transfer among pigments, each photosystem is ergodic and the configurational entropy is positive. This decreases the free energy of the single photosystem pigment array by a small amount. On the other hand, the suspension of photosystems is non-ergodic and the configurational entropy approaches zero. The overall configurational entropy which, in principle, includes contributions from both the single excited photosystems and the suspension which contains excited photosystems, also approaches zero. Thus the configurational entropy upon photon absorption by either a pigment solution or a suspension of photosystem particles is approximately zero.

Photochemical trapping heterogeneity as a function of wavelength, in plant photosystem I (PSI-LHCI).

In the present paper the marked changes in photochemical trapping time over the absorption/fluorescence band of isolated PSI-LHCI are studied by means of time resolved fluorescence decay measurements. For emission at 680-690nm the effective trapping time is close to 17-18ps, and represents the effective trapping time from the bulk antenna. At wavelengths above 700nm the effective trapping time increases in a monotonic way, over the entire emission band, to attain values in the range of 70-80ps near 760nm. This is argued to be caused by "uphill" energy transfer from the low energy states to the core antenna and reaction centre. These data, together with the steady state emission spectrum, permit calculation of the overall trapping time for maize PSI-LHCI, which is estimated to be approximately 40ps. The wavelength dependence of the trapping time indicates, that in PSI-LHCI there exists at least one red form which emits at lower energies than the 735nm state. These data indicate that Photosystem I is about 55% diffusion limited.

Functional analyses of the plant photosystem I-light-harvesting complex II supercomplex reveal that light-harvesting complex II loosely bound to photosystem II is a very efficient antenna for photosystem I in state II.

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.

The Q(y) absorption spectrum of the light-harvesting complex II as determined by structure-based analysis of chlorophyll macrocycle deformations.

The absorption spectrum of the main antenna complex of photosystem II, LHCII, has been modeled using, as starting points, the chlorophyll (chl) atomic coordinates as obtained by the LHCII crystal analysis [Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X., and Chang, W. (2004) Nature 428, 287-292] of three different trimers. The chl site Q(y) transition energies have been obtained in terms of the chl macrocycle deformations influencing the energy level of the chl frontier orbitals. Using these chl site transition energy values and the entire set of interaction energies, calculated in the ideal dipole approximation, the complete Hamiltonians for the three LHCII trimers have been written and the full set of 42 eigenstates of each LHCII trimer have been calculated. With the 42 transition energies and transition dipole strengths, either unperturbed or associated to the eigenstates, the LHCII Q(y) absorption spectrum has been calculated using a chl absorption band shape. These calculations have been performed both in vacuo and in the presence of a medium. Despite the number of approximations, a good correlation with the measured absorption spectrum of a LHCII preparation is obtained. This analysis shows that, although a substantial C3 symmetry of the LHCII trimer in terms of both chl-chl distances and interaction energies is present, a marked variation among monomer subsets of site transition energies is estimated. This leads to a C3 symmetry breaking in the unperturbed chl site transition energies set and, consequently, in the trimer eigenstates. It is also concluded that interactions among chlorophylls do not significantly modify the light absorption role of LHCII in plant leaves.

Band shape heterogeneity of the low-energy chlorophylls of CP29: absence of mixed binding sites and excitonic interactions.

A number of spectroscopic characteristics of three almost isoenergetic, red-shifted chlorophylls (chls) in the PS II antenna complex CP29 are investigated with the aim of (i) determining whether their band shapes are substantially identical or not, (ii) addressing the topical problem of whether they are involved in excitonic interactions with other chls, and (iii) establishing whether their binding sites may be defined as "mixed" with respect to their capacity to bind chls a and b. The three chls A2-CHL612, A3-CHL613, and B3-CHL614 were analyzed after in vitro apoprotein-pigment reconstitution using the CP29 coding sequence from Arabidopsis thaliana for both the wild-type and mutant complexes. Difference spectra thermal broadening analyses indicated that the half-bandwidths varied between 12 and 15 nm (at room temperature), due mainly to differences in the optical reorganization energy (25-40 cm(-1)). Moreover, only the A2 chl displayed an intense vibrational band in the 300-600 cm(-1) interval from the 0-0 transition. We conclude that within the red absorbing (approximately 680 nm) antenna chls of a single chl-protein complex a marked spectral band shape heterogeneity exists. By analysis of the absorption and circular dichroism spectra no evidence was found of significantly strong excitonic interactions. The single gene mutation of the A3 and B3 binding sites causes absorption changes in both the long wavelength chl a absorbing region and in the chl b spectral region. This has previously been observed and was attributed to "mixed" chl a/b binding sites [Bassi, R., Croce, R., Cugini, D., and Sandona, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96,10056-10061]. This interpretation, while in principle not being unreasonable, is shown to be incorrect for these two chls.

Fluorescence lifetime spectrum of the plant photosystem II core complex: photochemistry does not induce specific reaction center quenching.

The photosystem II kinetic model (diffusion or trap-limited) is still much debated. There is discussion about whether energy transfer from the core antenna (CP47 and CP43) to the reaction center complex (D1-D2-cyt b 559) is rate-limiting (transfer to trap-limited). This study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near 680 nm, due to P680 and an isoenergetic pheophytin. This was used as a marker feature for the reaction center complex. If the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near 680 nm, where there is reaction center complex specificity, than at the other emission wavelengths. Such a selective reaction center feature was not observed in fluorescence decay measurements. At the experimental resolution used here, we conclude that the trap-limited energy transfer to the reaction center could, at the most, be 20% limiting. Thus, the transfer to the trap-limited model is not supported. A kinetic, compartmental analysis was also performed on the data, taking into account a large number of separate measurements and the associated errors. Target analysis, considering these intermeasurement errors, yielded two minima which adequately describe the fluorescence lifetime data. The nonunique nature of the description is due to the fact that we have taken into consideration these intermeasurement errors. In our case, due to these errors, a correct kinetic model interpretation required additional experimental information.

On phytochrome absorption and the phytochrome photoequilibrium in a green leaf: environmental sensitivity and photoequilibrium time.

The average, corrected attenuance spectra for both spectral forms of phytochrome in a mature leaf were calculated. Optical masking by chlorophyll together with the detour effect (optical path lengthening effect) due to multiple light scattering led to large changes in both the Qy band shape and wavelength position and the effective intensity of the weak vibrational bands increases. The Pfr/Pr oscillator-strength-ratio between 400-750 nm (0.93 in vitro), becomes 1.63 in a leaf. Thus the dominant absorption form is Pfr. These two values permit calculation of the phytochrome photoequilibrium under conditions of "daylight" illumination both in vitro and in folia. These values are 0.6 and 0.38 respectively. Previous literature estimates for the situation in vitro, based on the 660/730 nm absorption ratio, yielded values close to 0.6. It is demonstrated that this large decrease in the phytochrome photoequilibrium in a leaf has the effect of translating this parameter to a position on the dose (red/far-red light ratio)-response (Pfr/Ptot) plot towards greater sensitivity to changes in the environmental red/far-red ratio. The increased sensitivity factor is almost five-fold for the "daylight" environment and is even greater for the various "shade-light" environments. The approximate time taken to attain photoequilibrium (1/e lifetime) has also been calculated for phytochrome in a leaf in different light environments. For the "daylight" environment the photoequilibration time is approximately 5 s, which increases into the 20-80 s interval under different degrees of "shade light". Thus, despite the strong optical masking by chlorophyll in a mature leaf, the phytochrome photoequilibrium is attained quite rapidly on a physiological time scale.

Entropy consumption in primary photosynthesis.

Knox and Parson have objected to our previous conclusion on possible negative entropy production during primary photochemistry, i.e., from photon absorption to primary charge separation, by considering a pigment system in which primary photochemistry is not specifically considered. This approach does not address our proposal. They suggest that when a pigment absorbs light and passes to an excited state, its entropy increases by hnu/T. This point is discussed in two ways: (i) from considerations based on the energy gap law for excited state relaxation; (ii) using classical thermodynamics, in which free energy is introduced into the pigment (antenna) system by photon absorption. Both approaches lead us to conclude that the excited state and the ground state are isoentropic, in disagreement with Knox and Parson. A discussion on total entropy changes specifically during the charge separation process itself indicates that this process may be almost isoentropic and thus our conclusions on possible negentropy production associated with the sequence of reactions which go from light absorption to the first primary charge separation event, due to its very high thermodynamic efficiency, remain unchanged.

Chlorophyll ring deformation modulates Qy electronic energy in chlorophyll-protein complexes and generates spectral forms.

The possibility that the chlorophyll (chl) ring distortions observed in the crystal structures of chl-protein complexes are involved in the transition energy modulation, giving rise to the spectral forms, is investigated. The out-of-plane chl-macrocycle distortions are described using an orthonormal set of deformations, defined by the displacements along the six lowest-frequency, out-of-plane normal coordinates. The total chl-ring deformation is the linear combination of these six deformations. The two higher occupied and the two lower unoccupied chl molecular orbitals, which define the Q(y) electronic transition, have the same symmetry as four of the six out-of-plane lowest frequency modes. We assume that a deformation along the normal-coordinate having the same symmetry as a given molecular orbital will perturb that orbital and modify its energy. The changes in the chl Q(y) transition energies are evaluated in the Peridinin-Chl-Protein complex and in light harvesting complex II (LHCII), using crystallographic data. The macrocycle deformations induce a distribution of the chl Q(y) electronic energy transitions which, for LHCII, is broader for chla than for chlb. This provides the physical mechanism to explain the long-held view that the chla spectral forms in LHCII are both more numerous and cover a wider energy range than those of chlb.

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii.

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

Photosynthesis research in Italy: a review.

This historical review was compiled and edited by Giorgio Forti, whereas the other authors of the different sections are listed alphabetically after his name, below the title of the paper; they are also listed in the individual sections. This review deals with the research on photosynthesis performed in several Italian laboratories during the last 50 years; it includes research done, in collaboration, at several international laboratories, particularly USA, UK, Switzerland, Hungary, Germany, France, Finland, Denmark, and Austria. Wherever pertinent, references are provided, especially to other historical papers in Govindjee et al. [Govindjee, Beatty JT, Gest H, Allen JF (eds) (2005) Discoveries in Photosynthesis. Springer, Dordrecht]. This paper covers the physical and chemical events starting with the absorption of a quantum of light by a pigment molecule to the conversion of the radiation energy into the stable chemical forms of the reducing power and of ATP. It describes the work done on the structure, function and regulation of the photosynthetic apparatus in higher plants, unicellular algae and in photosynthetic bacteria. Phenomena such as photoinhibition and the protection from it are also included. Research in biophysics of photosynthesis in Padova (Italy) is discussed by G.M. Giacometti and G. Giacometti (2006).

Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress.

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

Influence of the photosystem I-light harvesting complex I antenna domains on fluorescence decay.

The time-resolved fluorescence decay of plant PSI-LHCI has been analyzed and compared with its component parts, the PSI core and the peripheral antenna LHCI, in an attempt to (i) define the physical domains associated with the multicomponent decay-associated spectra (DAS) and determine the origin of the kinetically slow steps responsible for them, (ii) formulate a clear working hypothesis for the positive decay-associated spectral amplitudes of the two slowest decay components, and (iii) determine the impact of the peripheral antenna complexes (LHCI) on the effective trapping rate for the photosystem. The results for PSI-LHCI indicate that the three exponential component DAS description, previously reported in the literature, is not numerically unique. The fit minimum is rather broad, which necessitated the introduction of other fit criteria in addition to the purely numerical one. The analysis demonstrates that (i) the physical domains associated with the multicomponent decay are associated with the antenna and particularly with the low-energy spectral forms, (ii) the positive DAS amplitudes of the two slowest decay components are suggested to be due to energy transfer kinetic heterogeneity to different F735 low-energy forms, and (iii) the peripheral antenna slows down the effective photosystem photochemical rate by about 3 times, and this is approximately half due to antenna degeneracy and half due to the low-energy forms.

Photosynthesis and negative entropy production.

The widely held view that the maximum efficiency of a photosynthetic pigment system is given by the Carnot cycle expression (1-T/Tr) for energy transfer from a hot bath (radiation at temperature Tr) to a cold bath (pigment system at temperature T) is critically examined and demonstrated to be inaccurate when the entropy changes associated with the microscopic process of photon absorption and photochemistry at the level of single photosystems are considered. This is because entropy losses due to excited state generation and relaxation are extremely small (DeltaS << T/Tr) and are essentially associated with the absorption-fluorescence Stokes shift. Total entropy changes associated with primary photochemistry for single photosystems are shown to depend critically on the thermodynamic efficiency of the process. This principle is applied to the case of primary photochemistry of the isolated core of higher plant photosystem I and photosystem II, which are demonstrated to have maximal thermodynamic efficiencies of xi > 0.98 and xi > 0.92 respectively, and which, in principle, function with negative entropy production. It is demonstrated that for the case of xi > (1-T/Tr) entropy production is always negative and only becomes positive when xi < (1-T/Tr).

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation.

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.

The size of the population of weakly coupled chlorophyll pigments involved in thylakoid photoinhibition determined by steady-state fluorescence spectroscopy.

On the basis of experiments with singlet quenchers and in agreement with previous data, it is suggested that a population of energetically weakly coupled chlorophylls may play a central role in photoinhibition in vivo and in vitro. In the present study, we have used steady state fluorescence techniques to gain direct evidence for these uncoupled chlorophylls. Due to the presence of their emission maxima, near 650 nm and more prominently in the 670--675 nm interval both chlorophylls b and a seem to be involved. A straightforward mathematical model is developed to describe the data which allows us to conclude that the uncoupled/weakly coupled population size is in the range of 1--3 molecules per photosystem.

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I.

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.