|Title||Functional properties of the oxygen evolving complex of photosystem 2|
|Author(s)||Vliet, P.H. van|
|Source||Agricultural University. Promotor(en): T.J. Schaafsma; A.W. Rutherford. - S.l. : Van Vliet - ISBN 9789054855323 - 101|
|Publication type||Dissertation, externally prepared|
|Keyword(s)||fotosynthese - zuurstof - photosynthesis - oxygen|
This Thesis presents the results of a study by electron paramagnetic resonance (EPR) and measurements of oxygen evolution of the Oxygen Evolving Complex of Photosystem 11 (PS-II) in PS-II enriched membranes from spinach.
The experimental part of this Thesis is preceded by a general introduction (Chapter 1) and a brief overview and rationale of methods and techniques used (Chapter 2).
Chapter 3 describes an EPR study of PS-11 after Ca 2+depletion and subsequent Cl -depletion. The anions Ca 2+and Cl -are essential for oxygen evolution. After Ca 2+depletion in PS-II in the presence the Ca 2+chelator ethylene glycol his (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the S 2 state exhibits a modified multiline signal which is stable in the dark. It is found that the modification of S 2 is due to binding of EGTA to PS-11 which occurs after removal of Ca 2+. The pH buffer 4-(N-morpholino)ethanesulphonic acid (MES) modified the S 2 state in a similar fashion to EGTA as indicated by the EPR spectrum. EGTA and MES possibly bind with their anionic oxo-groups nearby or at the Mn cluster itself. It is also found that the EGTA binding- affinity is lowered by subsequent Cl -depletion in EGTA-treated/Ca 2+-depleted PS-II After Cl -depletion in the presence of millimolar EGTA concentrations, the S 2 state remains modified by bound EGTA. However, the S 2 state is not detected by EPR due to an additional modification of S 2 induced by Cl -depletion. Addition of Cl -in darkness reversed this Cl --depletion effect and resulted in the reconstitution of the EGTA-modified multiline EPR signal. Also the S 3 state is reversibly modified after Cl -depletion in Ca 2+-depleted PS-II resulting in a narrowing of the S 3 EPR signal. The Cl --dependent EPR properties of the S 2 and S 3 state in Ca 2+-depleted PS-II indicate that the Cl -which is essential for oxygen evolution, remains functionally bound after Ca 2+- depletion. The observed effects of Ca 2+and Cl -depletion in PS-II may he relevant to the proposed role(s) of Ca 2+and Cl -in controlling substrate binding in the charge accumulation cycle.
Chapter 4 presents an EPR study of the charge accumulation properties after Cl -depletion in PS-II whereas Ca 2+remains present in PS-II The light-intensity dependence of oxygen evolution is measured to study enzyme kinetics. The results indicate the presence of two Cl --binding sites in PS- II One of the sites is not essential for oxygen evolution and has not been previously reported in the literature. This site is depleted of Cl -by washes of PS-II membranes in Cl --free buffer solutions at pH 6.3. This Cl --depletion treatment results in a small decrease of the quantum yield of water oxidation and an increase of the S 2 g = 4 EPR signal intensity at the expense of the S 2 multiline EPR signal. The second site is essential for oxygen evolution and is equivalent to that studied in previous work on Cl --depleted PS-II. This site is depleted of Cl -by short incubation of diluted Cl --free washed PS-II membrane suspensions at pH 10. After this treatment no S 2 multiline signal can be generated and an intense S 2
Chapter 5 presents an EPR study of I --activated PS-II. The oxygen evolving activity of I --activated PS-II is nearly similar to that after Cl -reconstitution. A fraction of I --activated centers exhibits a characteristic S 2 g = 4 EPR signal. However, a second and significant fraction of active centers exhibits no S 2 EPR signal. The comparison with the effects of other anions described in Chapter 4 and in the literature, points to a correlation between the S 2 EPR properties and the size of the anion that occupies the Cl --site essential for oxygen evolution. The effects of I -on the properties of S 2 presumably reflect subtle structural changes of the Mn cluster since the I --induced modifications of S 2 are eliminated by addition of ethanol, resulting in the reconstitution of the normal S 2 multiline signal. However, no effects of ethanol are observed in pH 10/Cl --depleted and F --treated PS-II, both of which exhibit an intense g = 4 signal in the S 2 state (Chapter 4). This appears to indicate that the effects of ethanol on the S 2 EPR properties are modulated by the anion occupying the Cl -site essential for oxygen evolution. If the observed ethanol effects would originate from ethanol binding to PS-II the results may be relevant for the role of Cl -in the mechanism of water oxidation, and may indicate that Cl -modulates the substrate affinity.
In Chapter 6 the microwave power saturation of Tyr D.in untreated PS-II is investigated to reveal information on the magnetic properties of the oxygen evolving complex in the different oxidation states. The S 1 state is not detected by conventional EPR. Nevertheless, by using Tyr D.as a magnetic probe, two magnetically distinct forms of S 1 are detected which are interconverlible. After 30 min. dark-adaptation (0 °C) a rapidly relaxing S 1 Tyr D.is observed which is converted to a slowly relaxing form upon 17 h dark-adaptation (0 °C), in agreement with a pulsed EPR study in the literature. This conversion is accelerated by phenyl-p-benzoquinone (PPBQ) used as an electron acceptor. This effect of PPBQ is presumably is induced by the reduced form of PPBQ (PPBQH 2 ) since it is avoided by addition of relatively low concentrations of PPBQ to samples to which ferricyanide was added to maintain PPBQ in the oxidized form. It is shown that the slowly relaxing S 1 state becomes rapidly relaxing on the first enzyme cycle. The event responsible for this conversion occurs on the S 3 to S 0 transition or on the S 0 to S 1 transition. It has been previously proposed that the rapidly and slowly relaxing forms of S 1 correspond to a paramagnetic and diamagnetic S 1 state, respectively, reflecting structurally different Mn clusters. However, in view of the results from this work, it may be considered that the Mn cluster in S 1 is diamagnetic and that the rapidly relaxing TyrD. in S 1 is due to a nearby paramagnetic species different from the Mn cluster.
Chapter 7 presents a microwave power saturation study of Tyr D.in PS-II after Cl -depletion as described in Chapter 4. The spin state of the Mn cluster in Cl --depleted PS-II giving rise to an S 2 g=4 signal, significantly enhances the microwave power saturation of Tyr D.. However, on the basis of a mathematical model for the dipolar interaction between two spin systems, it is considered that the spins contributing to the S 2 g = 4 EPR signal are magnetically decoupled from Tyr D., due the mismatch between the g-values of the two spin systems. These results suggest that the S 2 g = 4 signal originates from an S = 3/2 spin state of the Mn cluster which also gives rise to a contribution of spins at g = 2.