Organization Of Photosystem Research Articles

Changes in the organization of Photosystem II following light-induced D 1-protein degradation

The composition and organization of photosystem II was studied in thylakoid membranes and subfractions which had been subjected to photoinhibitory light conditions. The results show that D 1-protein degradation can occur in vitro leading to a 50–60% loss of the protein. Apart from the D 2-protein, which shows a limited decline, there was no loss of any other photosystem II proteins. The D 1-protein degradation induced several changes in the organization of the photosystem II complex. Using inside-out thylakoid vesicles we demonstrate that concomitant with the D 1-protein degradation there is a release of the extrinsic 33, 23 and 16 kDa proteins from the inner thylakoid surface into the lumenal space. In addition, there is a release of four manganese ions per D 1-protein degraded. The correlation between the D 1-protein degradation and the release of manganese is also seen in inside-out thylakoid vesicles that have been CaCl 2-washed to remove the three extrinsic proteins prior to photoinhibitory illumination. Subfractionation of thylakoids subsequent to photoinhibitory treatment suggests a migration of certain photosystem II subunits from the appressed to the non-appressed thylakoid regions following D 1-protein degradation. The photosystem II subunits showed an individual migration behaviour, suggesting a disassembly of the photosystem II core. Our data suggest that repair of photodamaged photosystem II involves, apart from reinsertion of new D 1-proteins, reassembly of the photosystem II complex including lateral movement of proteins between the two thylakoid regions and religation of the manganese cluster.

Current perceptions of Photosystem II

In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.

Assembly of photosystem II polypeptides and expression of oxygen evolution activity in the chloroplasts of Euglena gracilis Z during the dark-light transition

Euglena cells ( Euglena gracilis Z) grown heterotrophically in the dark were illuminated, and the assembly of the polypeptides of Photosystem II in the thylakoid membrane during greening of the cells was studied by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western-blotting analysis with the antibodies against spinach Photosystem II polypeptides. In the dark-grown cells where the chloroplast is not developed, neither chlorophyll nor Photosystem-II-related polypeptide was detected. Upon illumination, chlorophylls and the polypeptides of Photosystem II began to be synthesized and accumulated in the thylakoid membrane. By comparing the amount of these membrane components in greening process of Euglena cells, it became evident that the extrinsic 30 kDa protein responsible for stabilization of Mn in oxygen-evolution system accumulated more slowly than the other components comprising the core complex of Photosystem II. In accordance with thiselectron transport activity from water to 2,6-dichloroin-dophenol in chloroplasts appeared later compared with that from 1,5-diphenylcarbazide to 2,6-dichloroindophenol, and the increase in the former activity was in parallel with the accumulation of the extrinsic 30 kDa protein in the thylakoid. These results suggest that accumulation and binding of the extrinsic 30 kDa protein to the core complex of Photosystem II is rate-limiting in the light-induced organization of Photosystem II and expression of the oxygen-evolution activity in Euglena . This feature is in contrast with that of higher plants and algae, where a considerable amount of extrinsic proteins of Photosystem II already exists in the dark growth condition and accumulation of the extrinsic proteins in the membrane itself is not a major determinant of the expression of oxygen evolution activity.

Effect of heptane-extraction on the stability of subunit organization of photosystem I reaction center complexes

Treatment of lyophilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. with n-heptane for 6 h resulted in marked changes in the pattern of photosystem I reaction center complexes resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis. CP1-a, which consists of two large subunits and three small subunits, was a major chlorophyll-containing band resolved from the lyophilized thylakoid membranes, whereas the heptane-extracted membranes produced mainly CP1-e which totally lacks the small subunits. Electron transport from the primary donor P700 to the secondary acceptor P430 was not affected by the heptane-extraction of the membranes. The heptane-treatment removed 97% of β-carotene present in the membranes, whereas all chlorophyll a, a major part of xanthophylls, more than a half of phylloquinone and one third of plastoquinone remained unextracted. The data suggest that β-carotene has an important structural effect to stabilize the subunit organization of photosystem I reaction center complexes but is not essential for the early photochemical events of photosystem I.

Effects of trypsin and bivalent cations on P-680+-reduction, fluorescence induction and oxygen evolution in Photosystem II membrane fragments from spinach

Laser-flash-induced absorption changes at 830 nm, fluorescence-induction curves and the average oxygen yield per flash have been measured in spinach Photosystem II membrane fragments as a function of trypsin treatment and its modification by CaCl2. The following was found. (i) The relative contribution of the nanosecond relaxation to the overall decay kinetics of 830 nm absorption changes reflecting the P-680+-reduction decreases as a function of incubation time with trypsin. Simultaneously, mild treatment at pH = 6.0 markedly increases the extent of 200 μs kinetics that highly revert back to nanosecond kinetics by CaCl2 addition. After harsher trypsin treatment (pH = 7.5) pH-dependent 2–20 μs kinetics appear that cannot be reverted to nanosecond kinetics by CaCl2. (ii) The CaCl2-induced restoration of nanosecond kinetics is mainly due to a Ca2+-induced effect rather than to a functional role of Cl−. Sr2+ can substantially substitute for Ca2+, whereas Mg2+, Mn2+ and monovalent ions are almost inefficient. (iii) A quantitative correlation between the extent of the nanosecond kinetics and the average oxygen yield per flash was not observed. (iv) If CaCl2 is present in the assay medium for trypsin treatment the samples are markedly protected to proteolytic degradation. This effect mainly refers to the reaction pattern of the acceptor side. Other bivalent cations can substitute Ca2+ for its protective function. (v) The CaCl2-induced protection to proteolytic attack is extremely sensitive to a very short trypsin pretreatment that does hardly affect the shape of the fluorescence induction curve. The results are discussed in relation to the functional and structural organization of Photosystem II.

Photochemical apparatus organization in the thylakoid membrane of Hordeum vulgare wild type and chlorophyll b-less chlorina f2 mutant

The stoichiometry of the electron-transport complexes and the chlorophyll (Chl) antenna size of the photosystems is correlated in chloroplasts from barley wild type and in the chlorophyll b-less chlorina f2 mutant. Chloroplasts from mutant plants displayed three characteristic changes in the organization of Photosystem I (PS I) and Photosystem II (PS II). (a) The light-harvesting antenna of PS II was lowered from about 250 Chl a + b in the wild type to only 50 Chl a molecules in the mutant. The light-harvesting antenna of PS I was lowered from about 185 Chl a + b in the wild type to about 150 Chl a molecules in the mutant. (b) The PS II PS I complex ratio was substantially higher, i.e., about 3.0, in the mutant vs. about 1.8 in the wild type. (c) Mutant chloroplasts lacked the differentiation of PS II into PS II α and PS II β. The enhanced PS II PS I ratio is explained as a response of the plant to the lowered light-harvesting capacity of PS II in the mutant chloroplasts. The apparent lack of PS II heterogeneity in the thylakoid membrane of the chlorina f2 correlates with the absence of the Chl a b light-harvesting complex II (LHC II) (Percival, M.P., Webber, A.N. and Baker, N.R. (1984) Biochim. Biophys. Acta 767, 582–589) and suggests a difference in the amount of LHC II associated with PS II α and PS II β.

Molecular organization of Photosystem II

Molecular organization of Photosystem II

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II.

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.

Structural organization of photosystem I reaction centers

Using a phosphoroscopic attachment to the dichrograph, light-induced circular dichroism spectra have been measured for chlorophyll-protein complexes of Photosystem I. Minor components at 672, 678 and 685 nm are observed in these spectra in addition to the components of dimer splitting of the P700 Q y transition at 691 and 698 nm. The minor components are due to the Chl672, Chl 678 and Chl 685 forms of antenna chlorophylls, the optical activity of which is changed 2–4% as a result of P700 oxidation. It is suggested that P700 is not an isolated dimer but that it is included in a local complex comprising 8–10 chlorophyll molecules with an exciton level splitting value of 120–140 cm −1.

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size, relative electron-transport capacity, and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II α and II β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (▵ A 320) and red (▵ A 700) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number ( N) of chlorophyll (Chl) molecules transferring excitation energy to PS II α, PS II β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N α of chlorophyll (Chl) molecules associated with PS II α was 234, while N β = 100 and N PS I = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II α and PS II β was analyzed on the basis of a core Chl a reaction center complex component and a Chl a b- LHC component. There is a dissimilar Chl a b- LHC composition in the three photosystems with 77% of total Chl b associated with PS II α only. The results indicate that PS II α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II β and PS I.

Electron donation to Photosystem II in reaction center preparations

The room-temperature EPR characteristics of Photosystem II reaction center preparations from spinach, pokeweed and Chlamydomonas reinhardii have been investigated. In all preparations a light-induced increase in EPR Signal II, which arises from the oxidized form of a donor to P-680 +, is observed. Spin quantitation, with potassium nitrosodisulfonate as a spin standard, demonstrates that the Signal II species, Z ⨥, is present in approx. 60% of the reaction centers. In response to a flash, the increase in Signal II spin concentration is complete within the 98 μs response time of our instrument. The decay of Z ⨥ is dependent on the composition of the particle suspension medium and is accelerated by addition of either reducing agents or lipophilic anions in a process which is first order in these reagents. Comparison of these results with optical data reported previously (Diner, B.A. and Bowes, J.M. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoyunoglou, G., ed.), Vol. 3, pp. 875–883, Balaban, Philadelphia), supports the identification of Z with the P-680 + donor, D 1. From the polypeptide composition of the particles used in this study, we conclude that Z is an integral component of the reaction center and use this conclusion to construct a model for the organization of Photosystem II.

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a II and X320 unless exogenous acceptors are present, which directly oxidize X320 −, such as K 3Fe(CN) 6. (c) In the presence of K 3Fe(CN) 6 the recovery kinetics of Chl a II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S 2 and S 3 in normal chloroplasts, significantly accelerates the recovery of Chl a II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D 1 to Chl a + II. ANT 2p simultaneously retards the decay kinetics of X320 − and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320 − and Chl a + II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D + 1 between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.

Structure and organization of System II photosynthetic units during the greening of a dark-grown Chlorella mutant

The formation and the organization of Photosystem II photosynthetic units during the greening of a dark-grown Chlorella vulgaris, mutant 5/520, have been investigated by analysing the kinetics of the “activation” of oxygen evolution and of the fluorescence induction. 1. 1. The existence during the early stages of the greening of a stationary photosynthesis demonstrates the presence of active Photosystem II at these initial stages, which are integrated in a functional whole, leading to overall photosynthesis. 2. 2. The rise-time of oxygen evolution has been measured using far-red and green light in order to estimate the relative number of chlorophylls per unit. The amount of chlorophyll a remains relatively constant during the greening, while the progressive addition of chlorophyll b causes the size of the units to increase approx. 2-fold. 3. 3. The induction kinetics of the fluorescence are exponential during the early phases of greening and later become distinctly sigmoidal; this suggests that the first units synthesized on the surface of the membrane are isolated from each other by obstacles preventing electronic excitation transfers and that such obstacles which might correspond to some distance between such units, can disappear at later stages, allowing energy transfers to occur. These observations suggest that the Photosystem II units represent organized functional entities. They apparently consist of a relatively constant number of chlorophyll a molecules, which during the greening is complemented progressively by the addition of chlorophyll b.