An investigation into the functional role of the D1:1 and D1:2 polypeptides in photosystem II in cyanobacteria : the effect of changing PSI/PSII ratio on photoinhibition in Synechococcus sp. PCC7942 /

The cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) adjusts its photosynthetic function by changing one of the polypeptides of photosystem II. This polypeptide, called Dl, is found in two forms in Synechococcus sp. PCC 7942. Changing the growth light conditions by increasing the light intensity to higher levels results in replacement of the original form of D 1 polypeptide, D 1: 1, with another form, D 1 :2. We investigated the role of these two polypeptides in two mutant strains, R2S2C3 (only Dl:l present) and R2Kl (only Dl:2 present) In cells with either high or low PSI/PSII. R2S2C3 cells had a lower amplitude for 77 K fluorescence emission at 695 nm than R2Kl cells. Picosecond fluorescence decay kinetics showed that R2S2C3 cells had shorter lifetimes than R2Kl cells. The lower yields and shorter lifetimes observed in the D 1 and Dl:2 containing cells. containing cells suggest that the presence of D 1: 1 results in more photochemical or non-photochemical quenching of excitation energy In PSII. One of the most likely mechanisms for the increased quenching in R2S2C3 cells could be an increased efficiency in the transfer of excitation energy from PSII to PSI. However, photophysical studies including 77 K fluorescence measurements and picosecond time resolved decay kinetics comparing low and high PSI/PSII cells did not support the hypothesis that D 1: 1 facilitates the dissipation of excess energy by energy transfer from PSII to PSI. In addition physiological studies of oxygen evolution measurements after photoinhibition treatments showed that the two mutant cells had no difference in their susceptibility to photoinhibition with either high PSI/PSII ratio or low PSI/PSII ratio. Again suggesting that, the energy transfer efficiency from PSII to PSI is likely not a factor in the differences between Dl:l and Dl:2 containing cells.

Comparison of two measures of PSI electron transport in winter rye

The number of P700 (the reaction centre of Photosystem I) converted to P700+, in winter rye, was determined by measuring the absorbance change at 820nm . It was found, with a single turnover flash, that thylakoids isolated from cold grown plants have a 50% greater number of P700 oxidized than thylakoids isolated from warm grown plants. Incubation of thylakoids in the dark at 35 C did not change the number of P700 oxidized. The conversion of P700 to P700+ with a single flash can be compared to a steady state rate of electron transport using a Clark electrode. The results for P700 oxidation using the absorbance change at 820 nm measure effects within the PSI complex whereas the results obtained from a Clark electrode measures steady state electron transport between the cytochrome blf complex and the PSI complex. In contrast to the results for P700 oxidation it was shown, using a Clark electrode, that both thylakoids from cold grown plants and thylakoids incubated at in the dark 35 C exhibited 50% higher rates of electron transport than thylakoids from warm grown plants. The correlation between the higher rate of steady state PSI electron transport observed in thylakoids isolated from cold grown winter rye and number of active PSI reaction centres localizes the site of the increase to the PSI reaction centre. In contrast the lack of correlation after incubation at 35 C indicates the increase in the rate of light saturated electron transport in thylakoids isolated from cold grown plants and thylakoids incubated in the dark at 35 C occur by different mechanisms.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Dictionary of the Zeta Psi Fraternity of North America

A hardcover Dictionary of the Zeta Psi Fraternity, edited by Arthur H. Motley and Harry B. Carpenter. An excerpt from the forward reads: "Having the sincere purpose of placing in the hands of all Brothers of the Fraternity a Directory of the full membership of our order, with addresses as perfectly correct as time and equipment would permit, supplemented with such alphabetical and geographical indices as will make the volume a handy and accurate cross-reference to our membership...". Included with this book is a typewritten letter from Gordon Waldie, treasurer of the Toronto Chapter of the Zeta Psi Fraternity thanking Mrs. Percy Band for the gift of the original initiation certificate of the late Hamilton K. Woodruff. Mr. Woodruff was one of the Charter members of the Fraternity and he was the 7th man initiated into this, the first Canadian chapter of any fraternity. The letter is dated Nov. 21, 1949. The full text is available in the Brock University Special Collections and Archives.

Characterization of the phosphorylation of thylakoid membrane proteins from cyanobacterium synechococcus sp. PCC 6301 in light state 1 and light state 2

The distribution of excitation energy between the two photosystems (PSII and PSI) of photosynthesis is regulated by the light state transition. Three models have been proposed for the mechanism of the state transition in phycobilisome (PBS) containing organisms, two involving protein phosphorylation. A procedure for the rapid isolation of thylakoid membranes and PBS fractions from the cyanobacterium Synechococcus m. PCC 6301 in light state 1 and light state 2 was developed. The phosphorylation of thylakoid and soluble proteins rapidly isolated from intact cells in state 1 and state 2 was investigated. 77 K fluorescence emission spectra revealed that rapidly isolated thylakoid membranes retained the excitation energy distribution characteristic of intact cells in state 1 and state 2. Phosphoproteins were identified by gel electrophoresis of both thylakoid membrane and phycobilisome fractions isolated from cells labelled with 32p orthophosphate. The results showed very close phosphoprotein patterns for either thylakoid membrane or PBS fractions in state 1 and state 2. These results do not support proposed models for the state transition which required phosphorylation of PBS or thylakoid membrane proteins.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

The measurement of the optical absorption cross section of photosystem 1 and photosystem 2 from whole live cells of porphyridium cruentum, in light state 1 and light state 2

The optical cross section of PS I in whole cells of Porphyridium cruentum (UTEX 161), held in either state 1 or state 2, was determined by measuring the change in absorbance at 820nm, an indication of P700+; the X-section of PS2 was determined by measuring the variable fluorescence, (Fv-Fo)/Fo, from PS2. Both cross-sections were 7 determined by fitting Poisson distribution equations to the light saturation curves obtained with single turnover laser flashes which varied in intensity from zero to a level where maximum yield occurred. Flash wavelengths of 574nm, 626nm, and 668nm were used, energy absorbed by PBS, by PBS and chla, and by chla respectively. There were two populations of both PSi and PS2. A fraction of PSi is associated with PBS, and a fraction of PS2 is free from PBS. On the transition S1->S2, only with PBS-absorbed energy (574nm) did the average X-section of PSi increase (27%), and that of PS2 decrease (40%). The fraction of PSi associated with PBS decreased, from 0.65 to 0.35, and the Xsection of this associated PS 1 increased, from 135±65 A2 to 400±300A2. The cross section of PS2 associated with PBS decreased from 150±50 A2 to 85±45 A2, but the fraction of PS2 associated with PBS, approximately 0.75, did not change significantly. The increase in PSi cross section could not be completely accounted for by postulating that several PSi are associated with a single PBS and that in the transition to state2, fewer PSi share the same number of PBS, resulting in a larger X-section. It is postulated that small changes occur in the attachment of PS2 to PBS causing energy to be diverted to the attached PSi. These experiments support neither the mobile-PBS model of state transitions nor that of spillover. From cross section changes there was no evidence of energy transfer from PS2 to PSi with 668nm light. The decrease in PS2 fluorescence which occurred at this wavelength cannot be explained by energy transfer; another explanation must be sought. No explanation was found for an observed decrease in PSi yield at high flash intensities.

Pigment orientation changes detected by low temperature linear dichroism spectroscopy: cold-hardening transition in phycobilisome-containing organisms

Low temperature (77K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. pee 6301, and cold-hardening in winter rye (Secale cereale L. cv. Puma). Samples were oriented for spectroscopy using the gel squeezing method (Abdourakhmanov et aI., 1979) and brought to 77K in liquid nitrogen. The linear dichroism (LD) spectra of Synechococcus 6301 phycobilisome/thylakoid membrane fragments cross-linked in light state 1 and light state 2 with glutaraldehyde showed differences in both chlorophyll a and phycobilin orientation. A decrease in the relative amplitude of the 681nm chlorophyll a positive LD peak was observed in membrane fragments in state 2. Reorientation of the phycobilisome (PBS) during the transition to state 2 resulted in an increase in core allophycocyanin absorption parallel to the membrane, and a decrease in rod phycocyanin parallel absorption. This result supports the "spillover" and "PBS detachment" models of the light state transition in PBS-containing organisms, but not the "mobile PBS" model. A model was proposed for PBS reorientation upon transition to state 2, consisting of a tilt in the antenna complex with respect to the membrane plane. Linear dichroism spectra of PBS/thylakoid fragments from the red alga, Porphyridium cruentum, grown in green light (containing relatively more PSI) and red light (containing relatively more PSll) were compared to identify chlorophyll a absorption bands associated with each photosystem. Spectra from red light - grown samples had a larger positive LD signal on the short wavelength side of the 686nm chlorophyll a peak than those from green light - grown fragments. These results support the identification of the difference in linear dichroism seen at 681nm in Synechococcus spectra as a reorientation of PSll chromophores. Linear dichroism spectra were taken of thylakoid membranes isolated from winter rye grown at 20°C (non-hardened) and 5°C (cold-hardened). Differences were seen in the orientation of chlorophyll b relative to chlorophyll a. An increase in parallel absorption was identified at the long-wavelength chlorophyll a absorption peak, along with a decrease in parallel absorption from chlorophyll b chromophores. The same changes in relative pigment orientation were seen in the LD of isolated hardened and non-hardened light-harvesting antenna complexes (LHCII). It was concluded that orientational differences in LHCII pigments were responsible for thylakoid LD differences. Changes in pigment orientation, along with differences observed in long-wavelength absorption and in the overall magnitude of LD in hardened and non-hardened complexes, could be explained by the higher LHCII monomer:oligomer ratio in hardened rye (Huner et ai., 1987) if differences in this ratio affect differential light scattering properties, or fluctuation of chromophore orientation in the isolated LHCII sample.

Electron Transfer Involving the Phylloquinone (A1) Cofactor of Photosystem I Examined with Time Resolved Absorbance and Electron Paramagnetic Resonance Spectroscopy

The dependence of the electron transfer (ET) rate on the Photosystem I (PSI) cofactor phylloquinone (A1) is studied by time-resolved absorbance and electron paramagnetic resonance (EPR) spectroscopy. Two active branches (A and B) of electron transfer converge to the FX cofactor from the A1A and A1B quinone. The work described in Chapter 5 investigates the single hydrogen bond from the amino acid residue PsaA-L722 backbone nitrogen to A1A for its effect on the electron transfer rate to FX. Room temperature transient EPR measurements show an increase in the rate for the A1A- to FX for the PsaA-L722T mutant and an increased hyperfine coupling to the 2-methyl group of A1A when compared to wild type. The Arrhenius plot of the A1A- to FX ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between A1A- and FX. The reasons for the non-Arrhenius behavior are discussed. The work discussed in Chapter 6 investigates the directionality of ET at low temperature by blocking ET to the iron-sulfur clusters FX, FA and FB in the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, by incorporating the high midpoint potential (49 mV vs SHE) 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the A1A and A1B binding sites. Various EPR spectroscopic techniques were implemented to differentiate between the spectral features created from A and B- branch electron transfer. The implications of this result for the directionality of electron transfer in PS I are discussed. The work discussed in Chapter 7 was done to study the dependence of the heterogeneous ET at low temperature on A1 midpoint potential. The menB PSI mutant contains plastiquinone-9 in the A1 binding site. The solution midpoint potential of the quinone measures 100 mV more positive then wild-type phylloquinone. The irreversible ET to the terminal acceptors FA and FB at low temperature is not controlled by the forward step from A1 to FX as expected due to the thermodynamic differences of the A1 cofactor in the two active branches A and B. Alternatives for the ET heterogeneity are discussed.