Saturating Flash Research Articles

Reversible, Water Stress‐Induced Non‐Uniform Chlorophyll Fluorescence Quenching in Wilting Leaves of Potentilla reptans may not be due to Patchy Stomatal Responses

Abstract: After exposure to full sunlight under natural conditions, attached leaves of the common meadow weed Potentilla reptans show non‐uniform (“patchy”) chlorophyll fluorescence quenching in the early stages of fluorescence transients. These areas of bright fluorescence can be readily reproduced in detached leaves that are allowed to wilt on the laboratory bench in weak light. The extent and duration of the patchiness increases with increasing water stress (higher relative saturation deficits). Images captured during saturating flashes show that the patches also display slow development of non‐photochemical quenching, consistent with the possibility that photosynthetic metabolism is impaired in these areas. Wilted Potentilla leaves readily regain full turgor when petioles are placed in water, and uniform chlorophyll fluorescence is recovered with in 30mm. Epidermal impressions reveal closed stomata over areas of both low and high fluorescence in wilted leaves. Because highly fluorescent patches also persist when wilted tissues are exposed to high CO2 (i.e., patchiness is unlikely to be due to local differences in CO2 supply) the data suggest direct effects of water stress on metabolism in wilted leaves. Leaf transverse sections show that although major veins may isolate areas of the lamina, minor veins do not. Relationships to leaf anatomy are discussed.

Photosynthetic characteristics of marine phytoplankton from pump-during-probe fluorometry of individual cells at sea.

Active fluorescence techniques are becoming commonly used to monitor the state of the photosynthetic apparatus in natural populations of phytoplankton, but at present these are bulk water measurements that average all the fluorescent material in each sample. Here we describe two instruments that combine individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence induction, on the time scale of 30 to 100 micros, with flow cytometric or visual characterization of each cell. In the PDP flow cytometer, we measure Chl fluorescence yield as a function of time during a 150 micros excitation flash provided by an argon ion laser; each particle is subsequently classified as in a conventional flow cytometer. In the PDP microfluorometer, individual cells in a sample chamber are visually identified, and fluorescence excitation is provided by a blue light-emitting diode that can be configured to provide a saturating flash and also a subsequent series of short flashlets. This sequence allows both saturation and relaxation kinetics to be monitored. Phytoplankton from natural samples and on-deck iron-enrichment incubation experiments in the Southern Ocean were examined with each PDP instrument, providing estimates of the potential quantum yield of photochemistry and the functional absorption cross section for photosystem 2, for either individuals (for cells larger than a few micrometers) or populations (for smaller cells). Results from initial field applications indicate that single-cell PDP measurements can be a powerful tool for investigating the nutritional state of phytoplankton cells and the regulation of phytoplankton growth in the sea.

P680+• Reduction Kinetics and Redox Transition Probability of the Water Oxidizing Complex as a Function of pH and H/D Isotope Exchange in Spinach Thylakoids

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH <6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH >7.0 values of <1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.

Flash fluorescence induction: a novel method to study regulation of Photosystem II

We demonstrate that arrays of light-emitting diodes can be used to generate single-turnover flashes of light that saturate Q A reduction in green algae. The fast version of a double-modulation fluorometer can measure induction during a single-turnover saturating flash (flash fluorescence induction). The method allows the effective antenna size, antenna heterogeneity and connectivity of Photosystem II to be measured without poisoning the organism with herbicide that blocks Q A − reoxidation. Using the same instrument, we have also measured the functional heterogeneity of Photosystem II by the fluorescence transient related to the flash-induced advancement of S-states in the oxygen-evolving complex.

Rapid light curves: A new fluorescence method to assess the state of the photosynthetic apparatus

Photosynthetic electron transport rates (ETR), calculated from chlorophyll fluorescence parameters, were compared in long term light and dark adapted as well as photoinhibited Pisum sativum leaves using a novel chlorophyll fluorescence method and a new instrument: rapid light curves (RLC) generated with the MINI-PAM. RLCs are plots of ETRs versus actinic irradiances applied for 10 s. Large changes in maximum electron transport rates (ETRmax) were observed when leaves were shifted from dark to moderate light, or from dark to photoinhibitory light and vice versa. Maximum ETRs were very low following long term dark adaptation, but increased to maximum levels within 8 to 15 minutes of illumination. It took more than 3 hours, however, to return irradiance-exposed leaves to the fully dark adapted state. Quenching analysis of RLCs revealed large qE development in long-term dark adapted leaves accounting for the low ETRs. Leaves photoinhibited for 3 hours had similarly reduced ETRs. In these leaves, however, qI was largely responsible for this reduction. Actinic irradiance exposures and saturating flashes affected leaves with different irradiance histories differently.

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z.

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.

S-state dependence of chloride binding affinities and exchange dynamics in the intact and polypeptide-depleted O2 evolving complex of photosystem II.

The Cl- binding properties in the successive oxidation states of the O2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl- can be replaced by NO3- in minutes, in an exchange reaction that depends on the NO3- concentration and that is not rate-limited by dissociation of Cl- from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO3- substitution reaction by an order of magnitude. A quantitative analysis of the Cl- concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl- binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S2 state than in the S1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl- binding site with the medium, but does not change the Cl- affinity of the binding site.

Catalase-free photosystem II: the O2-evolving complex does not dismutate hydrogen peroxide.

A photosystem II (PSII) membrane-associated heme catalase has been identified as a major source of the dark H2O2-dismutation reaction in PSII membrane samples [Sheptovitsky, Y. G., and Brudvig, G. W. (1996) Biochemistry 35, 16255-16263]. Based on this finding, a catalase-free PSII membrane sample was prepared by using mild heat treatment to deplete most of the PSII membrane-associated heme catalase followed by inhibition of the residual catalase with 50 mM 3-amino-1,2,4-triazole, a specific heme catalase inhibitor that binds covalently to compound I. After these treatments, the PSII membrane sample exhibited only 0.02% of the original H2O2-dismutation activity when assayed in the presence of 20 mM 3-amino-1,2,4-triazole. This small residual H2O2-dismutation activity is attributed to adventitious metal ions or the non-heme iron in PSII because the activity was still present in a Mn-depleted PSII sample but was completely suppressed by adding 5 mM ferricyanide to the assay buffer; the effect of ferricyanide is attributed to oxidation of H2O2-dismutating cations. Although the H2O2-dismutation activity was completely eliminated by these treatments, the light-induced O2-evolution activity was retained. A single saturating flash given to catalase-free PSII membranes did not induce any H2O2-dismutation activity. These results demonstrate that the S1/S-1 and S2/S0 cycles of the O2-evolving complex of PSII do not occur in the presence of H2O2, as proposed by Velthuys, B., and Kok, B. [(1978) Biochim. Biophys. Acta 502, 211-221]. The light-induced O2-evolution activity in catalase-free PSII was found to be irreversibly impaired by micromolar concentrations of H2O2. Thus, it is possible that the PSII membrane-associated heme catalase plays an important role in protection of the O2-evolving complex from damage by H2O2.

Kinetics of recovery of the dark-adapted salamander rod photoresponse.

The kinetics of the dark-adapted salamander rod photocurrent response to flashes producing from 10 to 10(5) photoisomerizations (Phi) were investigated in normal Ringer's solution, and in a choline solution that clamps calcium near its resting level. For saturating intensities ranging from approximately 10(2) to 10(4) Phi, the recovery phases of the responses in choline were nearly invariant in form. Responses in Ringer's were similarly invariant for saturating intensities from approximately 10(3) to 10(4) Phi. In both solutions, recoveries to flashes in these intensity ranges translated on the time axis a constant amount (tauc) per e-fold increment in flash intensity, and exhibited exponentially decaying "tail phases" with time constant tauc. The difference in recovery half-times for responses in choline and Ringer's to the same saturating flash was 5-7 s. Above approximately 10(4) Phi, recoveries in both solutions were systematically slower, and translation invariance broke down. Theoretical analysis of the translation-invariant responses established that tauc must represent the time constant of inactivation of the disc-associated cascade intermediate (R*, G*, or PDE*) having the longest lifetime, and that the cGMP hydrolysis and cGMP-channel activation reactions are such as to conserve this time constant. Theoretical analysis also demonstrated that the 5-7-s shift in recovery half-times between responses in Ringer's and in choline is largely (4-6 s) accounted for by the calcium-dependent activation of guanylyl cyclase, with the residual (1-2 s) likely caused by an effect of calcium on an intermediate with a nondominant time constant. Analytical expressions for the dim-flash response in calcium clamp and Ringer's are derived, and it is shown that the difference in the responses under the two conditions can be accounted for quantitatively by cyclase activation. Application of these expressions yields an estimate of the calcium buffering capacity of the rod at rest of approximately 20, much lower than previous estimates.

A sizeable decrease in the electric conductance of the thylakoid lumen as an early event during reaction center and Q cycle turnover.

A patch-clamp method was used for measuring light-induced currents (photocurrents) in single dark-adapted Peperomia metallica chloroplasts in a 'whole-thylakoid' configuration. The multi-phasic photocurrent profiles upon a train of multiple flashes (time separation between flashes in the train 1 s) show the following characteristics: (i) photocurrent generation originates from trans-thylakoid charge transfer accompanying reaction center (RC)- and Q-cycle turnover; (ii) a 15–30% decrease in the amplitude of the RC-driven current in the second and following flashes, concomitantly with an increase in the dark recovery time of the current; and (iii) a binary oscillation of the Q-cycle current generator with high activity in even numbered flashes. The decrease in amplitude and decay rate constant of the photocurrent in a double flash after dark adaptation are interpreted in terms of a change in the electric conductance of the thylakoid lumen. Data are interpreted to indicate a light control of the thylakoid lumen via a narrowing of the planar sheet-like structures by 1 to 3 single turnover flashes. A simple method is given to determine the bioenergetic and electric parameters of the thylakoid membrane of a single chloroplast from the current profiles in a double flash. The data indicate that 1 s after a saturating flash the fraction of closed inactive centers is less than 3%.

Different manganese binding sites in photosystem II probed by selective chemical modification of histidyl and carboxylic acid residues.

The binding of Mn2+ to manganese-depleted photosystem II was investigated after chemical modification of histidyl and carboxylic acid residues in the presence or absence of the native manganese cluster. K(M) values for Mn2+ were determined from steady-state electron transfer between Mn2+ and 2,6-dichlorophenolindophenol, the dissociation constant for Mn2+ was measured by observing the effect of added Mn2+ on the reduction of the primary donor P680+ after a saturating flash, and single-turnover electron donation from Mn2+ was followed by monitoring the decay kinetics of the EPR signal from the flash-induced tyrosine Zox radical. K(M) values for Mn2+ were found to be highly pH-dependent in both modified and unmodified photosystem II membranes. Treatment with histidine modifiers after removal of the manganese complex increased the K(M) values between 2.5 and 10 times and increased the dissociation constant for Mn2+ 8-fold, compared to membranes that were modified in the presence of the manganese cluster. Modification of carboxylic acid residues after removal of the manganese cluster increased the K(M) about 5-fold compared to membranes that were modified in the presence of the manganese cluster. The reduction rate of tyrosine Zox by Mn2+ was diminished after modification of either histidine or carboxylic acid residues. The apparent second-order rate constant decreased from 2.6 x 10(6) M(-1) s(-1) to 0.05 x 10(6) M(-1) s(-1) after histidine modification in the presence or absence of manganese, to 0.77 x 10(6) M(-1) s(-1) after carboxylic acid residue modification in the presence of manganese, and to 0.18 x 10(6) M(-1) s(-1) after carboxylic acid modification in the absence of manganese. Our results indicate the existence of two different manganese binding sites containing histidine, and at least two manganese sites with carboxylic acid residues, which are differently shielded against modifying agents by the native manganese cluster.

Phototransduction and calcium exchange along the length of the retinal rod outer segment.

The aim of this study was to investigate whether there are differences in calcium exchange between the ends of a retinal rod outer segment (OS) which could be correlated with the know differences of light responsiveness and which could explain the latter. We found that there is little, if any, difference in the exchange current density. However, the time constant of the exchange current decay after a saturating flash of light lengthened from base to tip and more calcium was extruded at the tip than the base of the OS. Our analysis of the results leads to the conclusion that there is little, if any, difference in free calcium concentration between the ends of the outer segment, but that there is a difference in the bound calcium and that this can explain the difference in light responsiveness along the length of the rod.

Ca2+ fluxes and channel regulation in rods of the albino rat.

By use of microelectrodes, changes in the receptor current and the Ca2+ concentration were measured in the rod layer of the rat retina after stimulation by flashes or steady light. Thereby light induced Ca2+ sources, and sinks along a rod were determined in dependence of time. Thus, the Ca2+ fluxes across the plasma membrane of a mammalian rod could be studied in detail. By light stimulation, Ca2+ sources are evoked along the outer segment only. Immediately after a saturating flash, a maximum of Ca2+ efflux is observed which decays exponentially with tau = 0.3 s at 37 degrees C (4.2 s at 23 degrees C). During regeneration of the dark current, the outer segment acts as a Ca2+ sink, indicating a restoration of the Ca(2+)-depleted outer segment. These findings agree with earlier reports on amphibian rods. Further experiments showed that the peak Ca2+ efflux and tau are temperature dependent. The peak amplitude also depends on the external Ca2+ concentration. In contrast to the reports on amphibian rods, only a part of the Ca2+ ions extruded from the outer segment is directly restored. Surprisingly, during steady light the Ca2+ efflux approaches a permanent residual value. Therefore, in course of a photoresponse, Ca2+ must be liberated irreversibly from internal Ca2+ stores. There is certain evidence that the inner segment acts as a Ca2+ store. Our results show that the Ca2+ fraction of the ions carrying the dark current is proportional to the extracellular Ca2+ concentration. This indicates that the Ca2+ permeability of the plasma membrane of the rod outer segment is independent of the Ca2+ concentration.

Changes in the expression of photosynthetic genes precede loss of photosynthetic activities and chlorophyll when glucose is supplied to mature spinach leaves

To study the molecular mechanism of ‘sink’ regulation on photosynthesis, thylakoid protein synthesis and content of photosynthetic proteins were compared with changes in photosynthetic activities and in leaf chlorophyll (chl) content in detached leaves kept on a diluted nutrient solution in the presence or absence of 50 mM glucose for 4 days at 60 μmol m −2 s −1. During the experiment, leaf chl content decreased in glucose-supplied leaves about 50%, in detached control leaves about 20%. The degradation of the photosynthetic apparatus was not connected with any substantial changes in photosynthetic parameters, when determined by pulse-modulated chl fluorescence at growth light conditions. However, at saturating light intensities of 1000 μmol m −2 s −1, leaf oxygen production was inhibited about 43% in leaves supplied with glucose, leading to a substantial increase in membrane energetization, as indicated by the increase of the non-photochemical quench, q n, and a down-regulation of photosystem (PS) II activity in the light, as determined by ΔF F′ m (yield of variable fluorescence under steady state conditions/fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions). No substantial changes were observed in the detached control leaves. The reduction of leaf oxygen production under external glucose supply was probably caused by a 60% loss of ribulose-1,5-bisphosphat-carboxylase, as revealed by Western blotting. In the detached control leaves, the content of Rubisco stayed stable. The content of D1 protein of the PS II reaction centre was reduced by 43% in the glucose-supplied leaves, when related to chl. However, a substantial loss of D1 protein of 30% was observed in the detached control leaves. As in other systems, the specific loss of D1 protein initiated the degradation of PS II and via the breakdown of light harvesting complex (LHC) II the loss of leaf chl. By measuring thylakoid protein synthesis, it could be shown that the changes in D1 protein content were caused by an inhibition of its synthesis, together with an increase in its degradation. In the glucose-supplied leaves, the expression of the 25 kDa polypeptide of the LHC II was also inhibited.

The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i.

A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S-modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s.

Static and dynamic actions of cytoplasmic Ca2+ in the adaptation of responses to saturating flashes in salamander rods.

1. In order to study the relative contribution to light adaptation of the various actions of Ca2+ in rod photoreceptors, changes in cytoplasmic calcium concentration ([Ca2+]i) were opposed by manipulating the calcium fluxes across the outer segment membrane at different times during the response to a bright flash. 2. When the outer segment was superfused with 0 Ca2+, 0 Mg2+,0 Na+ solution just before a bright flash, the period of response saturation was greatly prolonged. But if instead the solution change was made at progressively increasing times after the flash, the delay before the response recovered from saturation declined exponentially towards its value in Ringer solution with a time constant of around 1 s. In contrast, recovery time was little affected by stepping to 0 Ca+,0 Mg2+,0 Na+ solution before the flash and returning to Ringer solution shortly before the normal time of recovery from saturation. 3. When a bright flash was delivered just before the extinction of steady light, the response recovered from saturation progressively earlier as this steady intensity was increased. If, instead, the outer segment was transferred to 0 Ca2+,0 Mg2+,0 Na+ solution just before the bright flash then the time spent in saturation by the response was prolonged in darkness, but this additional delay progressively decreased as the steady intensity increased. 4. These results are consistent with the notion that the light-induced reduction of the time spent in saturation by the bright flash response in Ringer solution resulted from the static decrease in [Ca2+]i induced by the background, while the additional delay in the recovery from saturation when further changes in [Ca2+]i were prevented stemmed from the abolition of the dynamic fall in [Ca2+]i during the flash response. 5. Analysis of the effects of steady light on the time spent in saturation by the bright flash response under these conditions suggests that actions of [Ca2+]i at, or soon after, the time of the flash are largely responsible for the graded changes which take place in the bright flash response during light adaptation, while rapid actions of [Ca2+]i at the time of response recovery also play a role in the adaptation of the steady response to background light itself. 6. These data have been interpreted in terms of differential actions of [Ca2+]i on 'early' stages (e.g. events leading to phosphodiesterase activation) and 'late' stages (e.g. guanylyl cyclase) in the transduction mechanism. A quantitative model is presented which suggests that actions of [Ca2+]i on 'late' stages play a proportinately larger role in background adaptation than actions on 'early' stages.

TEMPERATURE DEPENDENCE OF CHLOROPHYLL(IDE) SPECTRAL SHIFTS and PHOTOACTIVE PROTOCHLOROPHYLLIDE REGENERATION AFTER FLASH IN ETIOLATED BARLEY LEAVES

Abstract— Absorbance spectroscopy at 77 K was used to investigate the effect of temperature on in vivo chlorophyllide shifts and photoactive protochlorophyllide regeneration after a saturating flash, which transformed all protochlorophyllide to chorophyllide. Photoactive protochlorophyllide present in darkness was stable up to 40°C. The rate of Shibata shift and protochlorophyllide regeneration after flash were strongly temperature dependent in the range 0–25°C. At 0°C, the shift was still observed but no regeneration occurred. Only slight effects were observed in the range 25–40°C. At all temperatures, the process of protochlorophyllide regeneration was significantly slower than the Shibata shift. The final chlorophyll shift from 672 to 674 nm was observed up to 40°C. The implication of these results concerning the pigment‐protein interactions during the Shibata shift are discussed.

Deletion of the PEST-like Region of Photosystem Two Modifies the QB-binding Pocket but Does Not Prevent Rapid Turnover of D1

The rapid turn-over of the D1 polypeptide of the photosystem two complex has been suggested to be due to the presence of a "PEST"-like sequence located between putative transmembrane helices IV and V of D1 (Greenberg, B. M., Gaba, V., Mattoo, A. K. and Edelman, M. (1987) EMBO J. 6, 2865-2869). We have tested this hypothesis by constructing a deletion mutant (delta 226-233) of the cyanobacterium Synechocystis sp. PCC 6803 in which residues 226-233 of the D1 polypeptide, containing the PEST-like sequence, have been removed. The resulting mutant, delta PEST, is able to grow photoautotrophically and give light-saturated rates of oxygen at wild type levels. However electron transfer on the acceptor side of the complex is perturbed. Analysis of cells by thermoluminescence and by monitoring the decay in quantum yield of variable fluorescence following saturating flash excitation indicates that Q-B, but not Q-A, is destabilized in this mutant. Electron transfer on the donor side of photosystem two remains largely unchanged in the mutant. Turnover of the D1 polypeptide as examined by pulse-chase experiments using [35S]methionine was enhanced in the delta PEST mutant compared to strain TC31 which is the wild type control. We conclude that the PEST sequence is not absolutely required for turnover of the D1 polypeptide in vivo although deletion of residues 226-233 does have an effect on the redox equilibrium between QA and QB.

Amino acid residues that influence the binding of manganese or calcium to photosystem II. 2. The carboxy-terminal domain of the D1 polypeptide.

To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate and histidine residues in the carboxy-terminal domain of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R.J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that His-332 Glu-333, His-337, and Asp-342 influence the assembly and/or stability of the manganese cluster, that His-332, Glu-333, and His-337 may ligate manganese, that Asp-342 may ligate manganese, calcium, or both, that Glu-333 and Asp-342 may play important structural roles, and that His-332, Glu-333, and His-337 influence the binding of calcium, although Glu-333 is unlikely to ligate Ca2+ directly. Several His-332, Glu-333, His-337, and Asp-342 mutants were very light sensitive, possibly because toxic activated oxygen species were released from altered or partly assembled manganese clusters. Finally, mutations at Asp-342 do not prevent posttranslational cleavage of the carboxy-terminal extension of the D1 polypeptide's precursor form in vivo.

Amino acid residues that influence the binding of manganese or calcium to photosystem II. 1. The lumenal interhelical domains of the D1 polypeptide.

To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or that are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate, histidine, and tyrosine residues in the lumenal interhelical domains of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R. J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that Asp-59 and Asp-61 may ligate calcium, that Asp-59, Asp-61, Glu-65, and His-92 influence the properties of the manganese cluster without significantly affecting its stability or ability to assemble, that Glu-189 plays an important structural role in maintaining the catalytic efficiency of the Mn cluster and partly influences the cluster's stability or ability to assemble, that His-92 and Glu-189 influence the binding of calcium, and that His-190 strongly influences the redox properties of the secondary electron donor, YZox, and either ligates manganese or serves as a crucial base or hydrogen bond donor. In addition, we conclude that Asp-170 may ligate manganese, but that its replacement with Val, Leu, or Ile causes structural perturbations that partly compensate for the loss of the carboxylate moiety.