Turnover kinetics of Photosystem I measured by the electrochromic effect in Chlorella

The rise kinetics of the absorption changes induced at 515 nm and 480 nm by a flash were studied using two types of xenon flashes of different durations. The ‘slow’ rise of the absorption change ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15–20 μ}\text{s}$) observed by Cox and Delosme (1978 C.R. Acad. Sci. (Paris) Sér. D 282, 775–778) and Joliot P., Delosme, R. and Joliot, A. ((1977) Biochim. Biophys. Acta 459, 47–57) was found to be due to double hits occurring in the reaction centers of System I during the flash.The turnover kinetics of the reaction centers of System I after a short flash were studied by a double flash method. They are in agreement with a second order reaction between P⁺-700 and its electron donor.     

Magnetic field effect on the chlorophyll fluorescence in Chlorella

The chlorophyll a fluorescence in Chlorella pyrenoidosa can be enhanced by 4–9% if the excitation light beam is parallel to an external magnetic field or decreased by 4–9% if the light beam is oriented perpendicular to a magnetic field of about 16 kgauss or more. These effects cannot be explained in terms of the small changes in light absorption which are also observed. It is suggested that these observations are due to a reorientation of pigment molecules in the magnetic field.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. I. Formation of System II photosynthetic units during greening under optimal light intensity

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a longer lag phase. (2) As a consequence, the chlorophyll: reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperativity between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase,cytochrome f and Photosystem II activity

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities.Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose.The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomonas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type.Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosystem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

Intensity effects on the fluorescence of in vivo chlorophyll

A technique for measuring relative quantum yields of fluorescence with a picosecond streak camera is described. We show that Chlorella pyrenoidosa exhibit an intensity dependent quantum yield when irradiated with single picosecond light pulses. This effect also occurs under conditions that inhibit the activity of the reaction centres, which can therefore be excluded as the cause.When a pulse train (pulse separation 6.9 ns) was used, the quantum yield was further reduced by the light absorbed from previous pulses, which indicates the formation of a quenching species having a relatively long lifetime.Absolute quantum yields calculated from the fluorescence decay show that single excitation pulses of 3 · 10¹³ photons/cm² give results comparable to those obtained by very low intensity methods.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Evidence for a double hit process in Photosystem II based on fluorescence studies

1. The amplitudes of the fast (0–20 μs) and slow (20 μs–2 ms) fluorescence rise induced by a 2 μs flash have been measured as a function of the energy of the flash in chloroplasts inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. The saturation curve for the slow rise shows a characteristic lag which is not observed for the fast fluorescence rise. This lag indicates that Photosystem II centers undergo a double hit process which implies that (a), each photocenter includes two acceptors Q₁ and Q₂; (b), after the first hit, oxidized chlorophyll Chl⁺ is reduced by a secondary acceptor Y in a time short compared to the duration of the flash; (c), after the second hit, Chl⁺ is reduced by another secondary donor, D.

2. According to Den Haan et al. ((1974) Biochim. Biophys. Acta 368, 409–421), hydroxylamine destroys the secondary donor responsible for the fast reduction of Chl⁺. In the presence of 3 mM hydroxylamine, only the secondary donor D is functional and a flash induces mainly a single hit process.

3. The saturation curves for the fast and the slow rises have been studied in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea for a second actinic flash given 2.5 s after a first saturating one. The large decrease in the half-saturating energy indicates the existence of efficient energy transfer occuring between photosynthetic units.

4. Two alternate hypotheses are discussed (a) in which D is an auxiliary donor and (b) in which D is included in the main electron transfer chain.     

Light inactivation of transaldolase in pea leaf chloroplasts

Transaldolase is inactivated when intact pea leaf chloroplasts are irradiated. This light inactivation is 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, arsenite and sulfite-sensitive, implicating the Light Effect Mediator system in light inactivation of this oxidative pentose phosphate pathway enzyme.     

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophyenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

A rapid,light-induced transient in electron paramagnetic resonance signal II activated upon inhibition of photosynthetic oxygen evolution

A rapid, light-induced reversible component in Signal II is observed upon inhibition of oxygen evolution in broken spinach chloroplasts. The inhibitory treatments used include Tris washing, heat, treatment with chaotropic agents, and aging. This new Signal II component is in a 1 : 1 ratio with Signal I (P700). Its formation corresponds to a light-induced oxidation which occurs in less than 500 μs. The subsequent decay of the radical results from a reduction which occurs more rapidly as the reduction potential of the chloroplast suspension is decreased. The formation of this free radical component is complete following a single 10-μs flash, and it occurs with a quantum efficiency similar to that observed for Signal I formation. Red light is more effective than far-red light in the generation of this species, and, in preilluminated chloroplasts, 3-(3,4-dichlorophenyl)-1,1-dimethylurea blocks its formation. Inhibition studies show that the decline in oxygen evolution parallels the activation of this Signal II component.These results are interpreted in terms of a model in which two pathways, one involving water, the other involving the rapid Signal II component, compete for oxidizing equivalents generated by Photosystem II. In broken chloroplasts this Signal II pathway is deactivated and water is the principal electron donor. However, upon inhibition of oxygen evolution, the Signal II pathway is activated.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

Determination and modification of the redox state of the secondary acceptor of Photosystem II in the dark

The redox state of the secondary electron acceptor B of Photosystem II was studied using fluorescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given $\text{B}\text{B}{}^{\mathrm{?}}$ ratio in the dark-adapted samples.We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in Chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts.Benzoquinone treatment modifies this distribution in Chlorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark.The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B^? is generated and stabilized in the dark.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Studies on the slow fluorescence decline in isolated chloroplasts

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar.At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission.Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.