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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.

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.

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.

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.

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.

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).

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.

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F(0) fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17+/-0.017 ns in the chlorina Photosystem II core to a value within the range 0.42+/-0.036-0.47+/-0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalized system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.

To investigate the light-harvesting properties of the Photosystem II chlorophyll (chl) a-b complexes (major light-harvesting complex of Photosystem II [LHCII], CP24, CP26, CP29) in a mature leaf under natural "daylight" illumination, the absorption spectra of the isolated complexes were converted into the photon absorption spectrum (1-T) within a leaf, using the approach of Rivadossi et al. ([1999] Photosynth. Res. 60, 209-215). In the Qy region, significant enhancement of light harvesting by the chl b electronic transitions, with respect to the absorption spectra (optical density [OD]), as well as a large and generalized increase (between two- and four-fold) associated with the vibrational bands of both chl a and b, was observed, which acquires an important light-harvesting role (approximately 30-40% of total). In the Soret region, a small increase in light harvesting by chl b was indicated. To gain more detailed information on these aspects the light harvesting of LHCII in a leaf was investigated. This required describing the pigment absorption (chl a and b, carotenoids) in the LHCII OD spectrum in terms of spectral subbands, which were subsequently used to estimate the relative light harvesting of each pigment type in LHCII of a leaf. When the entire visible spectral interval between 400 and 730 nm is considered, the chl a light harvesting is essentially unchanged with respect to the absorption spectrum (OD) of isolated LHCII, whereas the chl b contribution is 20% higher and the carotenoids are 33% lower. The relative enhancement of the chl b absorption is principally associated with the Qy electronic transition region, the light-harvesting contribution of which becomes prominent in the leaf.

The red antenna states of the external antenna complexes of higher plant photosystem I, known as LHCI, have been analyzed by measurement of their preequilibrium fluorescence upon direct excitation at 280 K. In addition to the previously detected F735 state, a hitherto undetected low-energy state with emission maximum around 713 nm was observed. The 280 K bandwidths (FWHM) are 55 nm for the F735 state and approximately 27 nm for the F713-nm state, much greater than for non-red-shifted antenna chlorophylls. The origin absorption band for the F735-nm state was directly detected by determination of its excitation (action) spectrum and lies at 709-710 nm. The absorption spectrum for F735, calculated using the Stepanov expression, closely overlaps the excitation spectrum, indicating that the very large Stokes shift (25 nm) is due to vibrational relaxation within the excited-state manifold and solvent effects can be excluded. Fluorescence anisotropy measurements, with direct excitation of F735, indicate that the transition dipoles of the two red states are parallel. Similar experiments performed in the long-wavelength absorbing tail of PSI-LHCI indicate the presence of emission state(s) that are red-shifted with respect to F735 of isolated LHCI. It is suggested that these are brought about by interactions between the complexes in PSI-LHCI, which occur in some yet undefined way, and which are broken upon solubilization of the component parts.

It has been previously demonstrated that the Stepanov equation provides a rather good description of the absorption/fluorescence spectra in Photosystem I, even though excited state equilibration is not rapid with respect to the excited state decay. In the present article this apparent contradiction is examined analytically for two-state systems and numerically for many-state systems. It is demonstrated that, in the special case of the trapping process being associated with the initially populated state, neither very rapid excited state equilibration nor a transfer equilibrium, which approximates a true Boltzmann distribution, are prerequisites to obtaining a very close approximation to a correct Stepanov result. This interesting conclusion is discussed in terms of plant Photosystem I (PSI-200). It is concluded that whereas, in compartmental modeling, photochemical trapping may be formally associated with the bulk antenna pigments due to the strong energy coupling between them and the trap pigments, this is not the case for the red spectral forms.

Selective excitation, at room temperature, in the long wavelength absorption tail of the photosystem I antenna complexes, known as light harvesting complex I, induces pronounced pre-equilibration fluorescence from the directly excited pigment state. This has allowed determination of the fluorescence band shape of this low energy photosystem I chlorophyll antenna state, at room temperature, for the first time. The emission maximum is near 735 nm. The remarkable band width (55 nm) and asymmetry have never been previously reported for chlorophyll a states.

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.

In the present study the analysis of the relation between the excited state population in the photosystem II (PSII) antenna and photoinactivation has been extended from an in vitro system, isolated thylakoids, to an in vivo system, Chlamydomonas reinhardtii cells. The results indicate that the excited state quenching by an added singlet quencher induces maximal protection against photoinhibition of about 30% of that expected on the basis of the observed light intensity-treatment time reciprocity rule. Similar results, obtained previously with thylakoids, have been interpreted in terms of damaged or incorrectly assembled complexes that play an important role in photoinhibition in the thylakoid membranes (Santabarbara, S., K. Neverov, F. M. Garlaschi, G. Zucchelli and R. C. Jennings [2001] Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids. FEBS Lett. 491, 109-113.). In an attempt to better define this aspect, the photoinhibition action spectra were determined for mutant barley thylakoids, lacking the chlorophyll (Chl) a-b complexes of the outer antenna, and for its wild type. The results indicate that in both systems the action spectra are significantly blueshifted (2-4 nm) and are broader than the PSII absorption in the membranes. These data are interpreted in terms of a heterogeneous population of outer and inner antenna pigment-protein complexes that contain significant levels of uncoupled Chl.

The room temperature absorption bandshape for the Q transition region of chlorophyll a is calculated using the vibrational frequency modes and Franck-Condon (FC) factors obtained by line-narrowing spectroscopies of chlorophyll a in a glassy (Rebane and Avarmaa, Chem. Phys. 1982; 68:191-200) and in a native environment (Gillie et al., J. Phys. Chem. 1989; 93:1620-1627) at low temperatures. The calculated bandshapes are compared with the absorption spectra of chlorophyll a measured in two different solvents and with that obtained in vivo by a mutational analysis of a chlorophyll-protein complex. It is demonstrated that the measured distributions of FC factors can account for the absorption bandshape of chlorophyll a in a hexacoordinated state, whereas, when pentacoordinated, reduced FC coupling for vibrational frequencies in the range 540-850 cm(-1) occurs. The FC factor distribution for pentacoordinated chlorophyll also describes the native chlorophyll a spectrum but, in this case, either a low-frequency mode (nu < 200 cm(-1)) must be added or else the 262-cm(-1) mode must increase in coupling by about one order of magnitude to describe the skewness of the main absorption bandshape.

In spinach thylakoids, the quenching of the singlet excited state in the photosystem II antenna by m-dinitrobenzene does not change the rate of the light induced degradation of the D1 reaction centre protein and offers only limited protection against photoinhibition itself. These results are discussed in terms of the role of non-photochemical quenching as a photoprotective strategy.

Selective destruction of the strongly dichroic red-shifted chlorophyll form (C709 nm) in photosystem I (PSI) trimers from Spirulina, by either non-selective high intensity illumination (photobleaching) or incubation with low concentrations of Triton X-100 is accompanied by changes in the circular dichroism spectrum of the same amplitude and of opposite sign at 677 nm. The data are interpreted in terms of a dimeric chlorophyll structure with excitonic bands at these two wavelengths. Similar photobleaching experiments with PSI-200 from maize also suggest the presence of bulk antenna/red form excitonic interactions.

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment-protein complexes may be involved.

In photosystem I trimers of Spirulina platensis a major long wavelength transition is irreversibly bleached by illumination with high-intensity white light. The photobleaching hole, identified by both absorption and circular dichroism spectroscopies, is interpreted as the inhomogeneously broadened Q(y) transition of a chlorophyll form that absorbs maximally near 709 nm at room temperature. Analysis of the mean square deviation of the photobleaching hole between 80 and 300 K, in the linear electron-phonon frame, indicates that the optical reorganization energy is 52 cm(-1), four times greater than that for the bulk, short-wavelength-absorbing chlorophylls, and the inhomogenous site distribution bandwidth is close to 150 cm(-1). The room temperature bandwidth, close to 18.5 nm, is dominated by thermal (homogeneous) broadening. Photobleaching induces correlated circular dichroism changes, of opposite sign, at 709 and 670 nm, which suggests that the long wavelength transition may be a low energy excitonic band, in agreement with its high reorganization energy. Clear identification of the 709-nm spectral form was used in developing a Gaussian description of the long wavelength absorption tail by analyzing the changing band shape during photobleaching using a global decomposition procedure. Additional absorption states near 720, 733, and 743 nm were thus identified. The lowest energy state at 743 nm is present in substoichiometric levels at room temperature and its presence was confirmed by fluorescence spectroscopy. This state displays an unusual increase in intensity upon lowering the temperature, which is successfully described by assuming the presence of low-lying, thermally populated states.

The spectral characteristics of fluorescence quenching by open reaction centres in isolated Photosystem II membranes were determined with very high resolution and analysed. Quenching due to photochemistry is maximal near 687 nm, minimal in the chlorophyll b emission interval and displays a distinctive structure around 670 nm. The amplitude of this 'quenching hole' is about 0.03 for normalised spectra. On the basis of the absorption spectra of isolated chlorophyll-protein complexes, it is shown that these quenching structures can be exactly described by assuming that photochemistry lowers the fluorescence yield of the reaction centre complex (D1/D2/cytb (559)) plus CP47, with quenching of the former complex being approximately double that of the latter complex. These data, which qualitatively indicate that there are kinetically limiting processes for primary photochemistry in the antenna, have been analysed by means of several different kinetic models. These models are derived from present structural knowledge of the arrangement of the chlorophyll-protein complexes in Photosystem II and incorporate the reversible charge separation characteristic of the exciton/radical pair equilibration model. In this way it is shown that Photosystem II cannot be considered to be purely trap limited and that exciton migration in the antenna imposes a diffusion limitation of about 30%, irrespective of the structural model assumed.

The red tail of the absorption spectrum of the D1-D2-cytb559 complex, defined as the absorption signal not described by the two Gaussian sub-bands associated with the intense electronic transitions at 680 and 683 nm, exhibits anomalous temperature behavior. This tail was analyzed in the temperature interval between 80 and 300 K in terms of the mean square deviation (sigma2) of the total Qy absorption band and by Gaussian sub-band decomposition. The value of the average optical reorganization energy (Snum) obtained from the temperature dependence of sigma2 for the whole absorption band was 32 cm(-1), and changed to 16-20 cm(-1) after subtraction of the sub-bands describing the red tail. This latter value is in agreement with the hole burning literature data for chlorophyll bound to proteins, and indicates that the rather high value for the apparent optical reorganization energy obtained by analysis of the total Qy band of the D1-D2-cytb559 complex is determined by the temperature sensitivity of the red tail. This suggests that the long wavelength absorption tail might be due to vibrational transitions associated with vibrational modes in the range of 80-150 cm(-1) which are thermally accessible and give rise to an absorption signal on the low-energy side of the (0,0) transition. On the basis of this assumption, the electron-phonon coupling strength (S) for these modes is estimated to be in the range 0.028-0.18. This interpretation furthermore supports the idea that the electronic transition near 683 nm is that of a monomer chlorophyll.

The photoinhibition-induced changes in Photosystem II fluorescence parameters of spinach thylakoids were only slightly sensitive to the excited state population in Photosystem II antenna, as modulated by either quinone quenching or energy spillover. The possibility that this may be due to a small fraction of chlorophyll molecules which are poorly coupled to the antenna is discussed.

The intact photosystem I of maize containing its full antenna complement (PSI-200) has been purified and fractionated into the core and outer antenna (LHCI) components. It is demonstrated by absorption and fluorescence spectroscopy that at least 80% of the long wavelength absorbing antenna pigments (red forms) are located in LHCI. Absorption spectra in the Qy region of all three preparations were measured between 72 and 300 K and subjected to a thermal broadening analysis. Data are interpreted in the linear electron-phonon coupling assumption, and the average optical reorganization energy (Snum) for the bulk pigment band and the red absorption tail determined. A marked asymmetry in Snum values across the absorption band is demonstrated. The bulk pigments in all three preparations have rather low values, in the range of 15-25 cm-1, suggesting that Stokes shifts for the absorption forms are in the 1. 5-3 nm range. On the other hand the red forms have markedly greater reorganization energies. While a direct thermal analysis of the red tail indicates minimum Snum values of around 60 cm-1, when the contribution of the red tail of the bulk pigments is corrected for in LHCI, the more reliable value of 110 cm-1 is obtained. These high Snum values for the red pigment forms suggest that they have unusually wide homogeneously broadened absorption bands and large Stokes shifts (6-11 nm).

The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

Absorption spectra in the Qy region of the light harvesting complex II (LHCII) have been measured in the temperature range 70-300 K. The spectra were analyzed by evaluating the temperature dependence (a) of the total bandwidth and (b) of the sub-bands obtained by numerical decomposition in terms of double Gaussians. The thermal broadening of the bands are interpreted, in both cases, as a homogeneous component, due to the presence of linear electron-phonon coupling, plus an inhomogeneous component, due to both statistical energy fluctuations at each pigment site and heterogeneity of the sample itself. Sub-bands analysis, in which eight major components are identified, yields a reorganization energy 9 cm(-1) < or = Svm < or = 14 cm(-1) and an inhomogeneous contribution in the range 120-170 cm(-1). In all cases the bands are substantially symmetrical in the 70-300 K temperature range. This observation gains theoretical support from an analysis of the band moments, when the influence of a low-frequency vibrational mode is considered. Analysis of the total absorption band yields Svm approximately 70 cm(-1); however, this high value is reduced to Svm approximately 11-20 cm(-1) when the red-most sub-band, with maximum at 684 nm, is eliminated at all temperatures. These data are discussed in terms of the underlying transitions, giving strong support to the presence of extreme red absorption bands in LHCII. The presence of another low-frequency mode with vm > 20-30 cm(-1) is also proposed.

Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-D-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < 200 K. A careful analysis of fluorescence between 75 and 280 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 720, 730, and 742 nm, the absorption origin bands of which have been calculated at 714, 725, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 dimer is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.26 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-2 ps-1.

Photosystem II, which has a primary photochemical charge separation time of about 300 ps, is the slowest trapping of all photosystems. On the basis of an analysis of data from the literature this is shown to be due to a number of partly independent factors: a shallow energy funnel in the antenna, an energetically shallow trap, exciton dynamics which are partly 'trap limited' and a large antenna. It is argued that the first three of these properties of Photosystem II can be understood in terms of protective mechanisms against photoinhibition. These protective mechanisms, based on the generation of non photochemical quenching states mostly in the peripheral antenna, are able to decrease pheophytin reduction under conditions in which the primary quinone, QA, is already reduced, due to the slow trapping properties. The shallow antenna funnel is important in allowing quenching state-protective mechanisms in the peripheral antenna.

Absorption spectra of the isolated D1/D2/cytochrome b-559 complex have been measured in the temperature range 80-300 K. All spectra were analyzed in terms of a linear combination of Gaussian bands and the thermal broadening data interpreted in terms of a model in which the spectrum of each pigment site is broadened by (a) a homogeneous component due to linear electron-phonon coupling to a low-frequency protein vibration and (b) an inhomogeneous component associated with stochastic fluctuations at each pigment site. In order to obtain a numerically adequate description of the absorption spectra, a minimum number of five sub-bands is required. Further refinement of this sub-band description was achieved by taking into account published data from hole burning and absorption difference spectroscopy. In this way, both a six sub-band description and a seven sub-band description were generated. In arriving at the seven sub-band description, the original five sub-band wavelength positions were essentially unchanged. Thermal broadening analysis of the seven sub-band description yielded data which displayed the closest correspondence with the literature observations. The wavelength positions of the sub-bands were near 661, 667, 670, and 675 nm, with two bands near 680 and 684 nm. The two almost isoenergetic sub-bands near 680 nm, identified as P680 and pheophytin, have optical reorganization energies around 40 and 16 cm-1, respectively. All other sub-bands, identified as accessory pigments, have optical reorganization energies close to 16 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of the absorption and fluorescence characteristics of the D1/D2/cytb-559 reaction centre complex of Photosystem II has been carried out by gaussian decomposition of absorption spectra both at room temperature and 72 K and of the room temperature fluorescence spectrum. A five component fit was found in which the absorption and fluorescence sub-bands could be connected by the Stepanov relation. The photobleaching and light-activated degradation in the dark of long wavelength pigments permitted a further characterisation of the absorption bands. The absorption (fluorescence) maxima of the five bands at room temperature are 660 nm (670 nm), 669 nm (675 nm), 675 nm (681 nm), 680 nm (683 nm), 681 nm (689 nm). A novel feature of this analysis is the presence of two approximately isoenergetic absorption bands near 680 nm at room temperature. The narrower one (FWHM=12.5 nm) is attributed to pheophytin while the broader band (FWHM=23 nm) is thought to be P680. The P680 band width is discussed in terms of homogeneous and site inhomogeous band broadening. The P680 fluorescence has a large Stokes shift (≈9 nm) and most fluorescence in the 690-700 nm range is associated with this chromophore.The three accessory pigment bands are broad (FWHM=17-24 nm) and the 660 nm gaussian is largely temperature insensitive thus indicating significant site inhomogeneous broadening.The very slight narrowing of the D1/D2/cytb-559 Qy absorption at crytogenic temperatures is discussed in terms of the coarse spectral inhomogeneity associated with the spectral forms and the apparently large site inhomogeneous broadening of short wavelength accessory pigments.

Room temperature and 10 K absorption and linear dichroism spectra of the chlorophyll-protein complexes comprising the outer antenna of PSII (LHCII, CP29, CP26, CP24) have been analyzed in terms of a linear combination of asymmetric Gaussian bands. The results demonstrate the following: (a) The absorption and linear dichroism spectra of each sample can be described by nearly the same set of Gaussian bands at room temperature and 10 K. (b) The relative distributions of the transition moments of the major red-absorbing spectral forms seem to be similar in all four outer antenna chlorophyll-protein complexes at room temperature, with the 684-nm band being oriented closest to the particle plane at room temperature and the 677- and 669-nm bands being tilted at progressively greater angles out of the particle plane. The shorter wavelength transitions seem to be oriented close to the magic angle, but interpretation is complicated in this spectral region due to the low linear dichroism values and by overlap with vibrational bands. (c) The 684-nm band, detected in room temperature absorption and linear dichroism spectra of all complexes, vanishes at 10 K.

The chlorophyll-protein complexes that form the antenna system of photosystem II have been purified and analyzed in terms of the commonly observed chlorophyll spectral forms. With the exception of chlorophyll b, which is known to be associated with the complexes comprising the outer antenna (LHCII, CP24, CP26, CP29), the spectral forms occur with similar absorption maxima and are present in rather similar amounts in each of the antenna complexes. On the basis of the published chlorophyll stoichiometries for the complexes in photosystem II antenna, the distribution of the spectral forms in a "reconstituted" antenna has been determined. These data were used to calculate the equilibrium population of excited states within the various chlorophyll-protein complexes within photosystem II. This was compared with the light absorption capacity of each of the complexes in the "reconstituted" antenna. The ratio of these two parameters (excited-state equilibrium distribution/absorption capacity) was determined to be 1.21 for the inner (core) antenna and 0.88 for LHCII. The standard free energy change for exciton transfer from the outer to the inner antenna was calculated to be -0.17 kcal mol-1. It is concluded that the photosystem II antenna is arranged as a very shallow funnel.

Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0-30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.

1. Chymotrypsin treatment of spinach chloroplast membranes does not change the electrophoretic mobility of either chlorophyll-protein complex 1 or 2. 2. The extraction of lipids with 80% acetone after treatment of the membranes with chymotrypsin reveals that the polypeptide components of both chlorophyll-protein complexes had been extensively digested. The extraction of carotenes with petroleum ether under the same conditions does not change the electrophoretic mobility of the chlorophyll-protein complexes. 3. Fluorescence polarisation studies of chlorophyll-protein complex 2 reveal that the chymotrypsin digestion of this complex does not result in changes of mutual orientation or distance apart of chlorophyll a, chlorophyll b or carotenoid. 4. Two polypeptide components have been detected after lipid extraction of electrophoretically purified chlorophyll-protein complexes 1 and 2. The SDS molecular weights are 24 000 and 27 000 for complex 2, and 68 000 and 64 000 for complex 1. 5. We conclude that chlorophyll performs an important structural function in both chlorophyll-protein complexes.

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent Km for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.

Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking.

The dependence of thylakoid osmotic volume on NH4Cl uncoupling and on phosphorylation substrates is determined by the centrifuge filtration method. The values obtained are used to evaluate the transmembrane proton gradient in conjunction with either the 9-aminoacridine fluorescence quenching method or the [14C]methylamine uptake method. The DeltapH values obtained with the two methods are compared and a linear relationship is demonstrated in the DeltapH range from 1.4 to 2.7 ([14C]methylamine values). Different linear relationships are obtained depending on the presence or absence of electron acceptor. We conclude that the 9-aminoacridine method can be used for DeltapH determination after calibration with other methods.

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl(2)) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl(2)) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the "diffuse electrical layer" hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar. At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission. Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sigmoidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116-125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.

An experimental analysis is presented concerning the effect on relative light absorption by the two photosystems caused by (a) a highly light scattering environment (the "detour effect") and (b) light filtration across successive chloroplast layers (the "light attenuation effect"). Both suspensions of isolated chloroplasts and leaves were employed.It is concluded that within a single spinach leaf these phenomena are likely to lead to only rather small increases in relative photosystem I absorption and activity with respect to photosystem II and will thus not exert a significant effect on non cyclic electron transport. On the contrary when light is filtrated across successive vegetation layers (shade light) significant increases in the relative PSI absorption and activity may be encountered.It is determined that the "detour effect" in mature leaves from a variety of plants increases overall photosynthetically useful light absorption by 35-40%.