Photosystem II reactions in oxidant-treated chloroplast fragments.

Treatment of Photosystem II fragments with the oxidant K₂IrCl₆ destroys approximately 50% of the bulk chlorophyll and results in fragments that are twofold enriched in P680 (the Photosystem II reaction-center chlorophyll) and cytochrome b₅₅₉. The fragments retain a fully competent reaction center, as evidenced by P680 photooxidation and subsequent reduction in a back reaction with the primary electron acceptor ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5}\text{ms}$ at 25 dgK). The K₂IrCl₆-treated fragments contain no photoactive or chemically detectable C-550 and do not exhibit any variable fluorescence. These results imply that the Photosystem II primary electron acceptor is unaffected by oxidant treatment. It therefore may be concluded that neither C-550 nor the fluorescence quencher, Q, functions as the primary electron acceptor of Photosystem II.     

Assignment of the low-temperature fluorescence in oxygen-evolving Photosystem II

Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b₅₅₉ subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a ‘slow-transfer’ pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 ‘slow-transfer’ states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680–700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

Picosecond laser study of fluorescence lifetimes in spinach chloroplast Photosystem I and Photosystem II preparations

Fractions enriched in either Photosystem I or Photosystem II have been prepared from chloroplasts with digitonin. A more detailed analysis of the decay kinetics of fluorescence excited by a picosecond laser pulse has been possible compared to experiments with unfractionated systems. The Photosystem I fractions show a very short component (? 100 ps) at room temperature which is apparently independent of pulse intensity over the range of photon densities used (5 · 10¹³–1 · 10¹⁶ photons cm^?2). The Photosystem II fraction has a short initial lifetime at room temperature which is strongly intensity-dependent approaching 500 ps at low photon densities, but decreasing to close to 150 ps at the highest photon densities. All of these room temperature decays appear to be non-exponential, and may possibly be fitted by at $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression, expected from a random diffusion of excitations via Förster energy transfer. On cooling to 77 K, lifetimes of both Photosystem I and Photosystem II increase, the lengthening with Photosystem I being more striking. The Photosystem I decays become intensity dependent like the Photosystem II, and at the lowest photon densities decays which are more nearly exponential within the experimental error give initial lifetimes of about 2 ns. The non-exponential decays seen at high photon densities appear to fit a $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression.     

S-state dependence of the miss probability in Photosystem II

The oxygen production of dark-adapted Photosystem II upon illumination by a series of single-turnover flashes shows a damped period four oscillation with flash number. The damping is attributed to `misses' resulting from a statistical probability that a reaction center fails to produce a stable charge separation after a saturating flash. The origin of misses is of interest because its probable dependence on flash number, in principle, affects the quantitative interpretation of all measurements on phenomena associated with the period four oscillation. We show that the kinetics of chlorophyll fluorescence yield transients induced by a flash series can be used to estimate the relative amplitudes of the miss probability on each flash. It is concluded that a major part of the misses must be caused by failure of the reduction of the oxidized primary electron donor chlorophyll P680⁺ by the secondary donor tyrosine Y_Z before the charge separation is lost by recombination. The probability of this failure is found to increase with the oxidation state of the oxygen-evolving complex: more than half of it occurs upon charge separation in the S₃ state, which is attributed to the presence of Y_Z ^ox S₂ in Boltzmann equilibrium with Y_ZS₃. This revised version was published online in June 2006 with corrections to the Cover Date.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

The relationship between the activity of chloroplast Photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with E_m7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, E_m7.8 = +383 mV, in addition to some of the low potential (E_m7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to ‘dark’ chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, E_m7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.     

Plastocyanin participation in chloroplast Photosystem I

French pressure cell disruption of spinach chloroplasts releases much of the plastocyanin from chloroplast membranes. Heavy particles obtained from French pressure cell disrupted chloroplasts lose most of their plastocyanin while light particles retain a high plastocyanin to chlorophyll ratio. Photosystem I activity is dependent on the presence of plastocyanin in our preparations.     

Isolation and characterization of chloroplast Photosystem II antenna of spinach by reversed-phase liquid chromatography

The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance liquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification has been accomplished by a combination of various SDS–PAGE systems employing either Comassie (or silver) staining or immunological detection using polyclonal antibodies raised against LHC II and against CP29, CP26 and CP24 proteins and by aminoacid microsequence. Moreover, peak identification is consistent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolution of all the protein components of the Photosystem II major Light Harvesting Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry in natural and stressed conditions, avoiding the expensive and time consuming separation procedure by sucrose-gradient ultracentrifugation and isoelectrofocusing.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed.At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a₆₈₀ and carotenoid pigments and a dampening of the sequence of O₂ evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination.Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.