The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

Detection of triplet states in algae by zero-field resonance.

Triplet states have been detected and characterized by zero-field splitting parameters in $\text{Anacystis nidulans}$, $\text{Euglena gracilis}$, $\text{Porphyridium cruentum}$, and $\text{Chlorella vulgaris}$, using fluorescence-detected magnetic resonance in zero-field at 4.2 K. Monitoring the 720 nm photosystem I emission, transitions between triplet spin levels have been assigned to antenna chlorophyll of one of both photosystems; photochemical reactions of chlorophyll are observed in the presence of an inhibitor and strong light, probably resulting in photoreduction and pheophytinization.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are $\text{D = 0.02794 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, $\text{E = 0.00382 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, i.e. equal to those of monomeric chlorophyll $\text{a}$ to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

Photoreduction of membrane bound cytochrome C by excited-state phenothiazine.

Ferricytochrome $\text{c}$ can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome $\text{c}$ which is adsorbed to the membrane surface. Under conditions when cytochrome $\text{c}$ is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome $\text{c}$, implying that the excited triplet state interacts with cytochrome $\text{c}$. Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome $\text{c}$ and phenothiazine is estimated to be over 20 Å.     

Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas speroides.

We report and discuss Raman spectra of bacteriochlorophyll $\text{a}$ and of bacteriopheophytin $\text{a}$ obtained $\text{in vitro}$ by resonance effect in their Q_X and Soret electronic bands. Selective excitation of spectra of either of these molecules in reaction centers of $\text{Rhodopseudomonas spheroides}$, strains Y and R 26, was achieved by illumination in their respective Q_X bands. Preliminary interpretation of the spectra yields information about the interactions assumed by these molecules in the reaction centers. Spheroidene bound to reaction centers of strain Y probably affects a conformation different from that assumed by the bulk spheroidene of the chromatophore.     

Spectral properties of a cyanobacterial cytochrome c oxidase: Evidence for cytochrome a.a3

Membranes isolated from $\text{Nostoc}$ sp. strain Mac oxidised NAD(P)H and horse heart ferrocytochrome c in dark reactions inhibited by KCN, NaN₃, CO, and by anaerobiosis. Reduced $\text{minus}$ oxidised difference spectra revealed peaks at 603 and 445 nm which shifted to 590 and 430 nm, respectively, in reduced $\text{plus}\text{CO}\text{minus}$ reduced spectra. In presence of suitable electron mediators the pigment could be reduced also with NAD(P)H or ascorbate; KCN prevented this reduction. Photoaction spectra of CO-inhibited membranes showed peaks at 590 and 430 nm. From the results it is concluded that cytochrome a.a₃ is a functional respiratory oxidase in $\text{Nostoc}$ sp. strain Mac.     

Photochemical oxidation of chlorpromazine in the dark induced by enzymically generated triplet carbonyl compounds

Enzymically generated triplet acetone and ethanal transfer energy to chlorpromazine as indicated by (i) suppression of the acetone chemiphosphorescence (ii) concomitant formation of chlo$\text{r}$ promazine photoproducts, that is the radical cation and the sulfoxide (iii) inhibition of photoproduct formation by a very efficient competition for triplet carbonyl energy using the sodium salt of 9,10-dibromoanthracene-2-sulfonic acid.This is the first report of a photooxidation in the dark.     

Fluorescence polarization study of human erythrocyte membranes with 1-phenyl-3-(2-naphthyl)-2-pyrazoline as orientational probe

The emission and polarization spectra of 1-phenyl-3-(2-naphthyl)-2-pyrazoline (PNP) in various environments were studied. Compared to the widely used orientational membrane probe 1,6-diphenylhexatriene (DPH), PNP is five times less photolabile and since its fluorescence emission maximum is at longer wavelengths $\text{(γ}{}_{\max }\text{≈ 445}\text{nm}\text{)}$, it is more suitable for use with intact erythrocytes. The limiting fluorescence anisotropy of PNP is 0.385. In erythrocyte ghosts, the steady-state emission anisotropy of PNP is a decreasing function of wavelength and its temperature dependence parallels that of DPH, dropping from 0.298 at 2°C to 0.185 at 38°C when averaged between 420 and 470 nm.     

ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester ($\text{I}$) and 1 : 14 ester ($\text{II}$). The nitroxide label of $\text{I}$ is aligned in the membrane with a very large angle of tilt (47° ± 1.5°) with respect to the normal to the membrane surface; $\text{II}$ does not show such a tilt. $\text{I}$ gives spectra corresponding to immobilized label while $\text{II}$ gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in $\text{I}$ and $\text{II}$ are not present at the phosphatidylcholine-water interphase.The data supports a ‘horseshoe’ configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.     

Membrane fluidity and drug metabolism in liver microsomes of lean, obob and dbdb mice

The thermally-induced changes of a cytochrome P-450 dependent activity (ethoxycoumarin-O-deethylase) and of the fluorescence polarization of diphenylhexatriene were compared in microsomes from lean, $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice. In lean mice, biphasic plots were obtained with break points in the same range of temperature by both methods, whereas, in $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice, no discontinuities were observed. These results may be related to a modified fatty acid composition of microsomal membranes in mutant mice. They exemplify the influence of the lipid environment on the monooxygenase system as also shown by the modified binding constants of cytochrome P-450 towards type II substrates in $\text{db}\text{db}$ mice.     

Photoreceptor pigment for blue light responses in Neurospora crassa

Irradiating the mycelium of $\text{Neurospora crassa}$ with blue light causes the photoreduction of a $\text{b}$-type cytochrome. The action spectrum for the photoreduction of the cytochrome $\text{b}$ is similar to action spectra for the photoactivation of carotene synthesis and photoinhibition of the circadian rhythm of conidiation in $\text{Neurospora}$.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Conformations and CD curves of cyclo(L- or D-Phe-L-Pro-Aca): cyclized models for specific types of β-bends

Cyclic tripeptides cyclo(L-Phe-L-Pro-Aca) (molecule $\text{3}$) (Aca, ?-aminocaproic acid) and cyclo(-D-Phe-L-Pro-Aca) (molecule $\text{4}$) are designed as models of specific types of β-bend. Energy calculation and ¹H and ¹³C NMR studies have indicated that peptides $\text{3}$ and $\text{4}$ form β-bend types VI and II', respectively. Circular dichroism spectra of $\text{4}$ have a double minimum negative band at the region of 200–230 nm like those of gramicidin S. The spectra of $\text{3}$, forming the cis peptide bond just before Pro, have a negative extremum at the 210–213 nm region. The spectra are used to estimate the contribution of various bend types in peptides.β-BendCD MeasurementConformational energy calculationCyclic peptideGramicidin SNMR measurement     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Magnetophotoselection applied to the triplet state observed by EPR in photosynthetic bacteria.

Magnetophotoselection (MPS) techniques have been used to study the triplet state observed by electron paramagnetic resonance (epr) in photosynthetic bacteria. Intact $\text{R.}\text{rubrum}$ chromatophores and systems in which the effects of energy transfer via antenna chlorophyll molecules have been minimized were examined. These preliminary results indicate that there is order in the bacteriochlorophyll antenna system, that the optical transition at 890 nm appears to be along the triplet y axis of the bacteriochlorophyll special pair (Bchl_sp), and that the resultant transition moment associated with 800 nm is approximately parallel to the long wavelength transition moment of the Bchl_sp.     

Protein-catalyzed phospholipid exchange in bilayer vesicles determined by flow cytometry and electron microscopy

The protein-induced lipid transfer between phosphatidylcholine vesicles was investigated. Measurements of the degree of polarization at single vesicles were made by flow cytometry using diphenylhexatriene as the optical probe. Vesicles differing in phase transition temperature could be distinguished by their degree of polarization at a temperature where one population was in the fluid ($\text{T > T}{}_{\mathrm{<mtext>t</mtext>}}$) and the other one in the quasi-crystalline ($\text{T < T}{}_{\mathrm{<mtext>t</mtext>}}$) state. Besides vesicles containing exchanged lipids we also observed fractions of unaffected vesicles. The lipid exchange was visualized directly by freeze-fracture electron microscopy. The characteristic ‘ripple’ structure of phosphatidylcholine vesicles disappeared upon exchange with lipid in the fluid state.     

Steric analysis of glycerophospholipids by circular dichroism. Stereospecifity of phospholipase D catalyzed transesterification.

The circular dichroism spectra of natural glycerophospholipids and synthetic 1-$\text{sn}$-phosphatidic acid were recorded. 3-$\text{sn}$-phosphatidic acid derivatives were found to show a positive Cotton effect, while 1-$\text{sn}$-phosphatidic acid revealed a negative Cotton effect. The results are interpreted in terms of the carboxyl sector rule. By this method phospholipase D was shown to produce stereospecifically 3-$\text{sn}$-phosphatidyl-1-$\text{sn}$-glycerol when incubated with egg yolk lecithin and exess of glycerol.     

Pulse fluorimetry of N-(1-pyrenesulfonyl)dipalmitoyl-l-α-phosphatidylethanolamine in concanavalin A-stimulated human lymphocytes

Human peripheral lymphocytes were cultured with a fluorescent probe, $\text{N-(1-}\text{pyrenesulfonyl)dipalmitoyl}\text{-}\text{l}\text{-α-}\text{phosphatidylethanolamine}$, and with concanavalin A. Fluorescence microscopic observations revealed that in lymphoblasts, pyrenesulfonyl dye was distributed mainly in vacuoles whereas in normal cells cultured without concanavalin A the dye was distributed exclusively in plasma membranes. The fluorescence spectra of the pyrenesulfonyl group incorporated into the cells exhibited two emission maxima, band A (monomer fluorescence of the pyrenesulfonyl group at about 400 nm) and band B (dimer fluorescence of the dye at about 500 nm). The values of the fluorescence lifetime measured at bands A and B indicated that in the absence of concanavalin A, the environment surrounding the pyrenesulfonyl group at the lipid/water interface became more hydrophilic with cultivation time. Concanavalin A made the environment of the interface more hydrophobic than that of lymphocytes cultured without concanavalin A. Fluorescence polarization measured at band A revealed that the mobility of pyrenesulfonyl monomers at the aqueous interface of the membranes was reduced upon concanavalin A stimulation.