THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES

Abstract
In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M N and N bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES*

Abstract— In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M → N and N → bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M → N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

THE EFFECT OF LEVULINIC ACID ON THE LIGHT INDUCED DEVELOPMENT OF PHOTOSYSTEM I AND II ACTIVITIES IN GREENING MAIZE LEAVES*

Photosystem I and Photosystem II activities were measured in chloroplasts isolated after 0–20 h illumination from etiolated maize leaves in which chlorophyll synthesis was specifically inhibited by levulinic acid. In control leaves not treated with levulinic acid, Photosystem I activity/chlorophyll developed rapidly during the first 2h in light, then fell off, and reached a constant level after 6h of illumination. In levulinic acid treated leaves, in which chlorophyll accumulation was inhibited up to 60%, a similar initial rise in Photosystem I activity was observed. However, the decrease in activity was much slower and continued for at least 20 h. The development of Photosystem I activity calculated on a leaf fresh weight basis was similar for control leaves or leaves treated with levulinic acid. This indicates that development of Photosystem I activity may not be related to chlorophyll accumulation during greening. Photosystem II activity/chlorophyll in leaves treated with or without levulinic acid increased similarly during the first 6h and then remained constant. Activity of Photosystem II per leaf fresh weight increased linearly, after the first h, for 20 h in the control leaves; in levulinic acid treated leaves this development was reduced by about 60%. Thus, development of Photosystem II activity can be related to chlorophyll accumulation. SDS gel electrophoresis of plastid membranes from control leaves illuminated for 12 h showed the presence of chlorophyll-protein complex I as well as Chl-protein 11; in the case of levulinic acid treated leaves only Chl-protein complex I was detectable, while Chl-protein complex II was markedly reduced.     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants dencient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.     

GENERATION AND PARTICIPATION OF O-2 IN 2-NITROPROPANE DIOXYGENASE REACTION

Abstract— 2-Nitropropane dioxygenase (EC 1. 13. 11) of the yeast Hansenula mrakii catalyzes the oxygenative denitrification of 2-nitropropane as follows:

The enzyme is significantly inhibited by superoxide dismutase and various scavengers for superoxide such as cytochrome c , epinephrine, thiols and polyhydric phenols. The scavengers added to the reaction mixture were oxidized or reduced. The addition of superoxide dismutase and the omission of 2-nitropropane or oxygen prevented the oxidation and the reduction of the scavengers. The enzyme catalyzes the formation of nitrite from 2-nitropropane by KO₂ added anaerobically.
One mole of NADH is bound per mole of the enzyme and predominantly the pro-R hydrogen of bound NADH is transferred to superoxide formed enzymatically or provided externally. The enzyme shows incomplete stereospecificity for hydrogen transfer from NADH.     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract— The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants deñcient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris–II. In vivo KINETICS OF THE DECAY OF Ii700 AND OF THE FORMATION OF Pfr STUDIED WITH A DOUBLE-LASER APPARATUS*

Abstract— Photoconversion of the red-light-absorbing form of phytochrome, P_r, to the far-red-light-absorbing form, P_fr, was investigated in vivo at 22°C with 600 or 800 ns laser pulses of high spectral purity and induction of spore germination in Dryopteris paleacea was used as indicator for the progress of photoconversion. This reaction is initiated by a saturating R-laser pulse of 648.5 nm, establishing an equilibrium of the photochromic system between P_r and the very early intermediates, Iⁱ₇₀₀ (P_rφ Iⁱ₇₀₀)- The decay of Iⁱ₇₀₀ as well as the formation of P_fr was recorded by the application of a second pulse varied between 698 and 717.5 nm, which inhibits the formation of P_lr being absorbed predominantly by Iⁱ₇₀₀or P_fr, respectively. The most effective inhibition for the second pulse is found up to 10 u.s after the first pulse and this is interpreted by photoreversion of Iⁱ₇₀₀ to P_r; thus reducing the formation of P_fr from Iⁱ₇₀₀. This early inhibition decreases between 10 μs to 100 ms after the R-laser pulse, as a result of the decay of Iⁱ_bl to a bleached species I,;. This decay can be described by three first order kinetics with the rate constants k₁₂= 16830 ± 2970 s^-1, k₁₂= 666 ± 218 s^-1,k₁₃= 9.8 ± 0.9 s^-1. A second inhibition, due to the formation of P_fr, is found for dark intervals <100 ms and can be described by two first order kinetics with the rate constants k₂₁= 2.9 ± 0.6 s^-1 and k₂₂= 0.17 s^-l.     

INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

KINETIC STUDIES ON THE STABILIZATION OF THE PRIMARY RADICAL PAIR P680+ Pheo- IN DIFFERENT PHOTOSYSTEM II PREPARATIONS FROM HIGHER PLANTS*

Abstract— The stabilization of the primary radical pair P680+ pheophytin (Pheo)^- through rapid electron transfer from Pheo to the special plastoquinone of photosystem II (PS II), Q_A, was analyzed on the basis of time-resolved (40 ps) UV-absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of¹Chi* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of ¹Chi* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of QA formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q^-_A formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q^-_A formation was found to be (300 ± 100 ps)^-1 in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.     

ANTAGONISTIC EFFECTS OF RED AND FAR-RED LIGHTS ON THE STABILITY OF PHOTOSYSTEM II IN PEA LEAVES EXPOSED TO HEAT

Abstract— The presence of light during exposure of intact pea leaves to high temperature (40°C) protects Photosystem II (PSII) against inactivation, as indicated by the preservation of the maximal variable 685 nm chlorophyll fluorescence and the photosynthetic oxygen evolution. This photoprotection was observed (i) to be saturated at low fluence rates ( ca 10 W m^-2) and (ii) to be strongly dependent on the spectral characteristics of the light. It was specifically induced by red light (630–670 nm) whereas other wavelengths were much less protective. A strong antagonism between red and far-red lights was also observed, with PSII stabilization by red light being partially cancelled by additional far-red light.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

ON THE NATURE OF THE INTERMEDIATE ELECTRON ACCEPTOR (A1) IN THE PHOTOSYSTEM I REACTION CENTER

Abstract— A sodium dodecyl sulfate-Photosystem I (PSI) complex has been prepared and characterized with respect to its electron acceptors. Component X and iron-sulfur centers A and B are absent from this preparation but the intermediate electron acceptor (A₁) is present. Flash-induced absorbance changes at 25°C show charge separation, followed by a back-reaction with a half-time of 5 µs. The spectrum of the flash-induced change from 350 to 550 nm indicates a contribution from the intermediate electron acceptor, A₁, as well as from P700⁺. EPR studies show that A₁ is associated with a free-radical signal having a g-value of 2.0025 and a linewidth of 12 gauss. A, would appear to be associated with a monomeric form of either Chi α or pheophytin a.     

EXTERNAL HEAVY ATOM EFFECTS ON THE S1 AND T1 ELECTRONIC STATES OF INDOLE

Abstract— The absorption, fluorescence and phosphorescence spectra along with the phosphorescence decay function of indole perturbed by various amounts of halocarbons have been studied in a 1:1 ethanol-ether glass at 77 K. For carbon tetrachloride. chloroform, halothane and methylene chloride used as quenchers. the biexponential nature of the phosphorescence decay prompted us to assume the existence of a triplet state complex (exciplex) between indole and the halocarbons. On the other hand. propyl bromide gives rise to a non-exponential decay and to an increase in the phosphorescence intensity suggesting the occurrence of a normal external spin-orbital coupling interaction between indole and the propyl bromide molecule.     

PHOTOCHEMICALLY INDUCED BINDING OF Rh(phen)2Cl2+ TO DNA†

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.