Quantum efficiency of native and mutant bacteriorhodopsin obtained from blue light induced relaxation experiments

Bacteriorhodopsin was continuously excited with green background light. In this way a steady state distribution of all intermediates of the photocycle was obtained. Then a perturbation of the system was induced by a blue laser flash and the resulting absorption changes were measured. The experiments were done with native bacteriorhodopsin and with the point mutant BR _Asp96Asn , in which aspartate 96 is changed to asparagine. Blue light induced relaxation experiments revealed a rate constant belonging to the excitation of bacteriorhodopsin by the green background. With this rate constant the quantum efficiency of native bacteriorhodopsin and of BR _Asp96Asn was determined to be 0.60 ± 0.10. Signals obtained with native bacteriorhodopsin could be explained with a simple model of the photocycle consisting of three consecutive intermediates BR ₅₆₈, L ₅₅₀ and M ₄₁₂. To describe the behavior of BR _Asp96Asn , a further photoactive intermediate after the M ₄₁₂ state had to be postulated. Properties of this intermediate are similar to those of the N ₅₅₀ state.     

Photocurrent kinetics of purple-membrane sheets bound to planar bilayer membranes

Summary The kinetics of light-driven proton transport by bacteriorhodopsin has been studied in a model system consisting of a planar lipid bilayer membrane to which purple membrane fragments have been attached. After excitation with a 10-nsec laser flash a fast negative current-transient occurs, followed by a positive transient which decays to zero. The time course of the photocurrent may be represented by a sum of four exponentials with time constants ₁= 1.2sec, ₂= 17sec, ₄= 57sec, ₁= 950sec (at 25°C). In a D₂O medium ₂ and ₃ are increased by a factor of 2.6 and 2.9, respectively, whereas ₁ remains unaffected. The observed components of the photocurrent can be correlated to photochemical reaction steps inferred from flash-photometric experiments on the basis of the observed time constants, the activation energies, and the effects of pH and D₂O. From the photocurrent signals information may be obtained on the magnitude of the charge displacement associated with the elementary transitions of the bacteriorhodopsin molecule.     

Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

Femtosecond and picosecond dynamics of recombinant bacteriorhodopsin primary reactions compared to the native protein in trimeric and monomeric forms

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bR_rec) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bR_rec was produced in an Escherichia coli expression system. Since bR_rec was prepared in a DMPC–CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bR_trim and bR_mon, respectively) was carried out. We found that bR_rec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bR_trim and bR_mon. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bR_rec, occurred more slowly compared to bR_trim, but similarly to bR_mon. The lifetime of intermediate I, judging from the signal of ΔA _ESA(470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bR_rec, 0.52 ps (73%) and 1.7 ps (27%) for bR_mon, and 0.45 ps (90%) and 1.75 ps (10%) for bR_trim. The formation time of intermediate K, judging from the signal of ΔA _GSA(625-635 nm), was 13.5 ps for bR_rec, 9.8 ps for bR_mon, and 4.3 ps for bR_trim. In addition, there was a decrease in the photoreaction efficiency of bR_rec and bR_mon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bR_rec are similar to those of the monomeric form of the native protein, bR_rec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.     

Inhibition of Photosystem 2 primary photochemistry by photogenerated protons

Photosystem 2 photochemical efficiency, measured as the rate of Qa reduction, was observed to be inhibited by preillumination with single turnover flashes, whilst F_o and F_m were not affected. Such inhibition was reversed by the uncoupler nigericin or by incubating the thylakoids in the dark for ca. 2 min after the preillumination. The presence of ATP in micromolar concentrations increased the time of dark recovery from the inhibition. The inhibition of fluorescence rise was not changed when 70% of the excitation energy available in the antenna was quenched by dinitrobenzene. Quantitative analysis of the observed fluorescence induction indicates that this phenomenon is due to the inhibition of the photochemical reaction itself. Uncouplers such NH₄Cl were unable to reverse the inhibition and only a few flashes of saturating intensity (10 or less) were required for the onset of it. This suggests that protons localised in domains rather than a pH gradient between the thylakoid lumen bulk solution and the external one are involved in this regulation of PS 2 efficiency.Abbreviations Chl- chlorophyll - cyt b ₅₅₉- cytochrome b ₅₅₉ - DCMU- 3-(3,4 dichlorophenyl)-1, 1 dimethylurea - DMBQ- dimethylbenzoquinone - DNB- dinitrobenzene - - electric potential difference - F_o- minimal fluorescence level measured with open reaction centres - F_m- maximal fluorescence level measured with closed reaction centres - F_v- variable fluorescence, defined as F_m-F_o - FWHM- full width at half maximum transmission - HA- hydroxylamine - MV- methylviologen - P680- pigment involved in the charge separation in Photosystem 2 - pheo- pheophytin - PS 1- Photosystem 1 - PS 2- Photosystem 2 - Q_a- primary quinone acceptor of Photosystem 2 - Q_b- secondary quinone acceptor of Photosystem 2     

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (q _{m, ap} ) and the apparent half-saturation concentration (K _ap ) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect q _{m, ap} , but K _ap is directly proportional to its concentration.Definitions for model parameters RX halogenated aliphatic substrate - E-M ⁿ reduced dehalogenase - E-M ⁿ⁺² oxidized dehalogenase - [E-M ⁿ ] steady-state concentration of the reduced dehalogenase (moles of reduced dehalogenase per unit volume) - [E-M ⁿ⁺²] steady-state concentration of the oxidized dehalogenase (moles of reduced dehalogenase per unit volume) - DH₂ primary exogenous electron-donor substrate - A primary exogenous electron-acceptor substrate - A2 second primary exogenous electron-acceptor substrate - X biomass concentration (biomass per unit volume) - f fraction of biomass that is comprised of the dehalogenase (moles of dehalogenase per unit biomass) - stoichiometric coefficient for the reductive dehalogenation reaction (moles of dehalogenase oxidized per mole of halogenated substrate reduced) - stoichiometric coefficient for oxidation of the primary electron donor (moles of dehalogenase reduced per mole of donor oxidized) - stoichiometric coefficient for oxidation of the endogenous electron donor (moles of dehalogenase reduced per unit biomass oxidized) - stoichiometric coefficient for reduction of the primary electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - stoichiometric coefficient for reduction of the second electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - r _RX rate of the reductive dehalogenation reaction (moles of halogenated substrate reduced per unit volume per unit time) - r _d1 rate of oxidation of the primary exogenous electron donor (moles of donor oxidized per unit volume per unit time) - r _d2 rate of oxidation of the endogenous electron donor (biomass oxidized per unit volume per unit time) - r _a1 rate of reduction of the primary exogenous electron acceptor (moles of acceptor reduced per unit volume per unit time) - r _a2 rate of reduction of the second primary electron acceptor (moles of acceptor reduced per unit volume per unit time) - k _RX mixed second-order rate coefficient for the reductive dehalogenation reaction (volume per mole dehalogenase per unit time) - k _d1 mixed-second-order rate coefficient for oxidation of the primary electron donor (volume per mole dehalogenase per unit time) - k _d2 mixed-second-order rate coefficient for oxidation of the endogenous electron donor (volume per mole dehalogenase per unit time) - b first-order biomass decay coefficient (biomass oxidized per unit biomass per unit time) - k _a1 mixed-second-order rate coefficient for reduction of the primary electron acceptor (volume per mole dehalogenase per unit time) - k _a2 mixed-second-order rate coefficient for reduction of the second primary electron acceptor (volume per mole dehalogenase per unit time) - q _m,ap apparent maximum specific rate of reductive dehalogenation (moles of RX per unit biomass per unit time) - K _ap apparent half-saturation concentration for the halogenated aliphatic substrate (moles of RX per unit volume) - k _ap apparent pseudo-first-order rate coefficient for reductive dehalogenation (volume per unit biomass per unit time)     

Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Magnesium binding to cation-depleted blue bacteriorhodopsin (b-bR) was studied spectrophotometrically as well as by following stopped-flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p-bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme

can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k₁, k₋₁ and k₂ are calculated to be 2.8 × 10⁴ M⁻¹ · s⁻¹, 5.0 × 10 s⁻¹ and 1 × 10⁻² s⁻¹, respectively.     

Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Mechanism of photoinhibition: photochemical reaction center inactivation in system II of chloroplasts

Photoinhibition of photosynthesis is manifested at the level of the leaf as a loss of CO₂ fixation and at the level of the chloroplast thylakoid membrane as a loss of photosystem II electron-transport capacity. At the photosystem II level, photoinhibition is manifested by a lowered chlorophyll a variable fluorescence yield, by a lowered amplitude of the light-induced absorbance change at 320 nm (A₃₂₀) and 540-minus-550 nm (A_540–550), attributed to inhibition of the photoreduction of the primary plastoquinone Q_A molecule. A correlation of the kinetics of variable fluorescence yield loss with the inhibition of Q_A photoreduction suggested that photoinhibited reaction centers are incapable of generating a stable charge separation but are highly efficient in the trapping and non-photochemical dissipation of absorbed light. The direct effect of photoinhibition on primary photochemical parameters of photosystem II suggested a permanent reaction center modification the nature of which remains to be determined.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Linear dichroism of single crystals of the reaction center from Rhodobacter sphaeroides wild type strain 2.4.1

The linear dichroism of single crystals of the photochemical reaction center from Rhodobacter sphaeroides 2.4.1, expressed as the anisotropy (or polarization) ratio, p = (A – A )/A + A , relative to the long morphological axis of the crystals, has been measured to be –0.12±0.03 for the primary donor Q _y and -0.15±0.8 for the carotenoid. These dichroic effects can be predicted using data obtained from magnetophotoselection (Frank et al. 1979, McGann and Frank 1985) and electron spin resonance (ESR)(Frank et al. 1988a, Budil et al. 1988) experiments. Magnetophotoselection data yield the projections of the transition moments onto the primary donor triplet state principal magnetic axis system. The single crystal triplet state ESR experiments provide the Euler matrix for the transformation from the principal magnetic axis system to the crystal unit cell axis system. Thus, the projections of the transition moments (site 1) onto the crystal units cell axes (a, b, c) are determined to be-0.39, 0.90 and 0.18, respectively. The projections of the carotenoid transition moment (site 1) onto the crystal unit cell axes (a, b, c) are determined to be -0.60, 0.02 and 0.80, respectively. This information used in conjunction with the crystalline space group symmetry (P₂₁2₁2₁) and the morphology of the crystals allows one to predict the observed anisotropy ratios.     

Spectral hole burning of the primary electron donor state of Photosystem I

Persistent photochemical hole burned profiles are reported for the primary electron donor state P700 of the reaction center of PS I. The hole profiles at 1.6 K for a wide range of burn wavelengths (_B) are broad (FWHM310 cm^-1) and for the 45:1 enriched particles studied exhibit no sharp zero-phonon hole feature coincident with _B. The _B hole profiles are analyzed using the theory of Hayes et al. [J Phys Chem 1986, 90: 4928] for hole burning in the presence of arbitrarily strong linear electron-phonon coupling. A Huang-Rhys factor S in the range 4–6 and a corresponding mean phonon frequency in the range 35–50 cm^-1 together with an inhomogeneous line broadening of100 cm^-1 are found to provide good agreement with experiment. The zero-point level of P700^* is predicted to lie at710 nm at 1.6K with an absorption maximum at702 nm. The hole spectra are discussed in the context of the hole spectra for the primary electron donor states of PS II and purple bacteria.Abbreviations NPHB nonphotochemical hole burning - O.D. optical density - PSBH phonon sideband hole - PS I Photosystem I P680 - P700, P870, P960 the primary electron donors of Photosystem II, Photosystem I, Rhodobacter sphaeroides, Rhodopseudomonas viridis - PED primary electron donor - RC reaction center - ZPH zero-phonon holes     

A computer modeling postulated mechanism for angiotensin II receptor activation

The angiotensin II receptor of the AT₁-type has been modeled starting from the experimentally determined three-dimensional structure of bacteriorhodopsin as the template. Intermediate 3D structures of rhodopsin and ₂-adrenergic receptors were built because no direct sequence alignment is possible between the AT₁ receptor and bacteriorhodopsin. Docking calculations were carried out on the complex of the modeled receptor with AII, and the results were used to analyze the binding possibilities of DuP753-type antagonistic non-peptide ligands. We confirm that the positively charged Lys¹⁹⁹ on helix 5 is crucial for ligand binding, as in our model; the charged side chain of this amino acid interacts strongly with the C-terminal carboxyl group of peptide agonists or with the acidic group at the 2-position of the biphenyl moiety of DuP753-type antagonists. Several other receptor residues which are implicated in the binding of ligands and the activation of receptor by agonists are identified, and their functional role is discussed. Therefore, a plausible mechanism of receptor activation is proposed. The three-dimensional docking model integrates most of the available experimental observations and helps to plan pertinent site-directed mutagenesis experiments which in turn may validate or modify the present model and the proposed mechanism of receptor activation.     

Comparative ecophysiological effects of drought on seedlings of the Mediterranean water-saver Pinus halepensis and water-spenders Quercus coccifera and Quercus ilex

Ecophysiological and structural traits of seedlings of the water-saver Pinus halepensis and the water-spenders Quercus coccifera and Q. ilex were studied in response to water stress under greenhouse conditions. Water deficit reduced stomatal conductance (g _s) and, as a consequence, both net CO₂ assimilation (A) and transpiration rate (E) were also reduced. Water stress also emphasized midday down-regulation of the photochemical efficiency (dynamic photoinhibition) reducing quantum yield of noncyclic electron transport (Φ_PSII), photochemical quenching (qP) and photochemical efficiency of the open reaction centres of PSII () and involved an increase of thermal dissipation of excess energy. However, water stress not only induced dynamic photoinhibition but also brought a reduction in F _v/F _m (chronic photoinhibition). Despite the water-saving strategy of P. halepensis that limited net CO₂ assimilation, this species showed a higher photochemical efficiency and lower photoinhibition than Quercus species. This was not the result of a different photochemical quenching but was linked to a higher value of , indicating a less severe photo-inactivation of PSII. Water stress resulted in a loss of pigment content and in an increase of the carotenoids/chlorophyll ratio, antioxidant capacity and the biomass rate allocated to roots as opposed to that assigned to leaves. P. halepensis showed a lower photoinhibition and antioxidant activity than Quercus species due to its lower pigment content and higher proportion of carotenoids allowing P. halepensis to use, in a more effective way, the lesser excess energy absorbed.     

A model of the phototactic reaction chain of the cyanobacterium Anabaena variabilis

The hypothesis has been proposed that in Anabaena variabilis the phototactic reaction sign is regulated by an unknown reaction sign reversal generator which is controlled by the intracellular level of singlet molecular oxygen (¹O₂). This hypothesis is supported by the following findings presented in this paper: Gassing with N₂ and Ar shifts the phototactic transition point at which the positive reaction becomes negative to higher fluence rates. Surprisingly this is true also for gassing with molecular oxygen ³O₂. Since ¹O₂ is produced in photosynthesis, the availability of external molecular oxygen seems not to be important. Apparently, a stream of any gas which is fast enough to remove ¹O₂ from the surface of the Anabaena trichomes decreases the internal ¹O₂ concentration and this way acts on the reaction sign reversal generator. Moreover, several carotenoids such as the water-soluble crocetin and preparations of solubilized -carotene, canthaxanthine and the C₃₀-ester ethyl--apo-8-carotenoate shift the transition point of phototaxis to higher fluence rates by about one order of magnitude. Several tested furan derivatives, such as dimethylfuran, diphenylisobenzofuran, and furfuryl ethanol, are either cytotoxic or not water-soluble at the concentrations necessary for an effective ¹O₂ quenching. Based one these results a model of the phototactic reaction chain of A. variabilis is proposed.Abbreviations DABCO 1,4-diazabicyclo(2.2.2)octane - DMF dimethylfuran - DPBF diphenylisobenzofuran Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

Effect of trypsin on D1/D2-cytochrom b 559 Photosystem 2 reaction center complex and reaction center from Rhodopseudomonas viridis

Proteolytic enzyme (trypsin) was used to structurally alter the RCs isolated from plant and bacterium as a way of probing the relation between structure (chromophore-apoprotein interactions) and function (photochemical activity). It was found that neither spectral characteristics (absorption spectrum, the 4th derivative of absorption spectrum) nor photochemical activity (pheophytine photoreduction, P680 photooxidation, etc.) were changed dramatically in D₁/D₂/cytochrom b ₅₅₉ PS 2 reaction center complex digested with trypsin. The PS 2 RC treated with trypsin migrates by one green band during electrophoresis with dodecylmaltoside. The peptides with a molecular mass higher than 3–4 kDa were not separated from PS 2 RC. These data indicate that digestion of D1 and D2 proteins does not disturb yet the conformation of peptides or their interactions in so-called core of RC and the native state of pigments. In contrast to that, the RC from Rhodopseudomonas viridis treated with enzyme has changed absorption spectrum and lost photochemical activity. The stability of the bacterial RC increased after exchange of LDAO by dodecylmaltoside.Abbreviations Chl chlorophyll a - Cyt cytochrome - DPC diphenylcarbazide - Dodecylmaltoside dodecyl--D-maltoside - LDAO lauryldimethylamino oxide - Pheo pheophytine - PS 2 Photosystem 2 - RC reaction center - SiMo silicomolybdate - SD sodium dodecyl sulfate     

Three-dimensional structures of photosynthetic reaction centers

In this article, the three-dimensional structures of photosynthetic reaction centers (RCs) are presented mainly on the basis of the X-ray crystal structures of the RCs from the purple bacteria Rhodopseudomonas (Rp.) viridis and Rhodobacter (Rb.) sphaeroides. In contrast to earlier comparisons and on the basis of the best-defined Rb. sphaeroides structure, a number of the reported differences between the structures cannot be confirmed. However, there are small conformational differences which might provide a basis for the explanation of observed spectral and functional discrepancies between the two species.A particular focus in this review is on the binding site of the secondary quinone (Q_B), where electron transfer is coupled to the uptake of protons from the cytoplasm. For the discussion of the Q_B site, a number of newlydetermined coordinate sets of Rp. viridis RCs modified at the Q_B site have been included. In addition, chains of ordered water molecules are found leading from the cytoplasm to the Q_B site in the best-defined structures of both Rp. viridis and Rb. sphaeroides RCs.Abbreviations B_A accessory bacteriochlorophyll in the active branch - B_B accessory bacteriochlorophyll in the inactive branch - D primary electron donor (special pair) - D_L special pair bacteriochorophyll bound by the L subunit - D_M special pair bacteriochorophyll bound by the M subunit - Q_A primary electron acceptor quinone - Q_B secondary electron acceptor quinone - RC reaction center - Rb. Rhodobacter - Rp. Rhodopseudomonas - _A bacteriopheophytin in the active branch - _B bacteriopheophytin in the inactive branch     

Reduced levels of cytochrome b 6/f in transgenic tobacco increases the excitation pressure on Photosystem II without increasing sensitivity to photoinhibition in vivo

We have examined tobacco transformed with an antisense construct against the Rieske-FeS subunit of the cytochromeb ₆ f complex, containing only 15 to 20% of the wild-type level of cytochrome f. The anti-Rieske-FeS leaves had a comparable chlorophyll and Photosystem II reaction center stoichiometry and a comparable carotenoid profile to the wild-type, with differences of less than 10% on a leaf area basis. When exposed to high irradiance, the anti-Rieske-FeS leaves showed a greatly increased closure of Photosystem II and a much reduced capacity to develop non-photochemical quenching compared with wild-type. However, contrary to our expectations, the anti-Rieske-FeS leaves were not more susceptible to photoinhibition than were wild-type leaves. Further, when we regulated the irradiance so that the excitation pressure on photosystem II was equivalent in both the anti-Rieske-FeS and wild-type leaves, the anti-Rieske-FeS leaves experienced much less photoinhibition than wild-type. The evidence from the anti-Rieske-FeS tobacco suggests that rapid photoinactivation of Photosystem II in vivo only occurs when closure of Photosystem II coincides with lumen acidification. These results suggest that the model of photoinhibition in vivo occurring principally because of limitations to electron withdrawal from photosystem II does not explain photoinhibition in these transgenic tobacco leaves, and we need to re-evaluate the twinned concepts of photoinhibition and photoprotection.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlophenyl)-1,-dimethylurea - Fo and Fo minimal fluorescence when all PS II reaction centers are open in dark- and light-acclimated leaves, respectively - Fm and Fm maximal fluorescence when all PS II reaction centers are closed in dark- and light-acclimated leaves, respectively - Fv variable fluorescence (Fm-Fo) in dark acclimated leaves - Fv variable fluorescence (Fm-Fo) in lightacclimated leaves - NPQ non-photochemical quenching of fluorescence - PS I and PS II Photosystem I and II - P680 primary electron donor of the reaction center of PS II - PFD photosynthetic flux density - Q_A primary acceptor quinone of PS II - q_p photochemical quenching of fluorescence - V+A+Z violaxanthin+antheraxanthin+zeaxanthin     

Reduction of N2 by Fe2+ via Homogeneous and Heterogeneous Reactions

Because of their high kinetic barrier and thermodynamic favorability under moderately reducing atmospheric composition photochemical approaches appear to be ideally suited to the direct reduction of solvated nitrogen gas. Ferrous iron has been investigated as an electron donor, as it has a highly tunable redox character and is environmentally ubiquitous. Recent advances in mineralogy connected to the field of environmental remediation have led to the identification of an important class of rusts which have reductive potentials comparable to Fe(0). These materials possess redox couples that are, potentially, capable of overcoming the kinetic barrier to the production of NH₃. In this study, we attempted to produce ammonia from N₂ by oxidizing white rust both photochemically and in a dark reaction. All results indicated the reaction was inhibited by competing reactions; primarily the reduction of H₂O to H₂. However, the dark reactions showed limited potential for reduction up to 1.4 mM. As a result, we turned to the question of closure temperature; the minimum temperature of rapid reaction based on a choice of reductant, which we demonstrate a model for its estimation. Due to the high thermodynamic energy of the intermediate, we conclude that aqueous photochemical reduction under the conditions studied here is an unlikely prebiotic source for reactive, i.e. reduced, nitrogen.     

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Summary The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate substites are required for the preferred substrates. Cl^– and HCO ₃ ^– , to account for tight binding in the transition state (K _diss1m). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unsual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and tunneling, which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.     

Temperature dependence of cytochrome photooxidation and conformational dynamics of Chromatium reaction center complexes

A temperature dependence of multiheme cytochrome c oxidation induced by a laser pulse was studied in photosynthetic reaction center preparations from Chromatium minutissimum. Absorbance changes and kinetic characteristics of the reaction were measured under redox conditions where one or all of the hemes of the cytochrome subunit are chemically reduced (E _h =+300 mV or E _h =–20 to -60 mV respectively). In the first case photooxidation is inhibited at temperatures lower than 190–200 K with the rate constant of the photooxidation reaction being practically independent on temperature over the range of 300 to 190 K (k=2.2×10⁵ s^-1). Under reductive conditions (E _h =–20 to -60 mV) lowering the temperature to 190–200 K causes the reaction to slow from k=8.3×10⁵ s^-1 to 2.1×10⁴ s^-1. Under further cooling down to the liquid nitrogen temperature, the reaction rate changes negligibly. The absorption amplitude decreases by 30–40% on lowering the temperature. A new physical mechanism of the observed critical effects of temperature on the rate and absorption amplitude of the multiheme cytochrome c oxidation reaction is proposed. The mechanism suggests a close interrelation between conformational mobility of the protein and elementary electron tunneling act. The effect of freezing conformational motion is described in terms of a local diffusion along a random rough potential.