Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

Evidence for variable chlorophyll fluorescence of photosystem I in vivo

Photosynthesis Research - Room temperature fluorescence in vivo and its light-induced changes are dominated by chlorophyll a fluorescence excited in photosystem II, F(II), peaking around...     

Wavelength-dependent photodamage to <Emphasis Type="Italic">Chlorella</Emphasis> investigated with a new type of multi-color PAM chlorophyll fluorometer

A new type of multi-color PAM chlorophyll fluorometer (Schreiber et al. 2012) was applied for measurements of photodamage to photosystem II (PS II) in optically thin suspensions of Chlorella (200 μg Chl l^?1) in the presence of 1 mM lincomycin. An action spectrum of the relative decrease of F _v/F _m in the 440–625 nm range was measured, which not only showed the expected high activity in the blue, but at a lower level also substantial activity above 540 nm. With the same dilute suspension, a PS II absorption spectrum was derived via measurements of the O-I₁ rise kinetics induced by differently colored strong light at defined incident quantum flux densities. After normalization of the two spectra at 625 nm, the relative extent of photodamage at 440–480 nm proved substantially higher than absorption by PS II, whereas the two spectra were close to identical in the 540–625 nm range. Hence, overall photodamage to PS II appears to consist of two components, one of which is due to light absorbed by PS II pigments, whereas the other one is likely to involve direct light absorption by Mn in the oxygen-evolving complex (Hakala et al. Biochim Biophys Acta 1706:68–80, 2005). Based on this rationale, an action spectrum of the Mn mechanism of photodamage was deconvoluted from the overall action spectrum, declining steeply above 480 nm. An almost identical Mn-spectrum was derived by another approach with the PAR of the various colors being adjusted to give identical rates of PS II turnover, PAR _II. The tentative, basic assumption of negligibly small contribution of the Mn mechanism to photodamage above 540 nm was supported by supplementary measurements using an external 665 nm lamp. 665 nm not only gave about two times PS II turnover as compared to 625 nm, but also about two times photodamage.     

Continuous ECS-indicated recording of the proton-motive charge flux in leaves

Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO₂ uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO₂ concentrations, except for very low CO₂, where the rate of charge flux distinctly exceeded the rate of CO₂ uptake. Parallel oscillations in CO₂ uptake and charge flux were induced by high CO₂ and O₂. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.     

Rapidly reversible chlorophyll fluorescence quenching induced by pulses of supersaturating light in vivo

The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100–400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm′, induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.

     

Light-induced dynamic changes of NADPH fluorescence in Synechocystis PCC 6803 and its ndhB-defective mutant M55

Blue-green fluorescence emission of intact cells of Synechocystis PCC6803 and of its ndhB-defective mutant M55 was measured with a standard pulse-amplitude-modulation chlorophyll fluorometer equipped with a new type of emitter-detector unit featuring pulse-modulated UV-A measuring light and a photomultiplier detector. A special illumination program of repetitive saturating light pulses with intermittent dark periods (10 s light, 40 s dark) was applied to elicit dynamic fluorescence changes under conditions of quasi-stationary illumination. The observed effects of artificial electron acceptors and inhibitors on the responses of wild-type and mutant M55 cells lead to the conclusion that changes of NAD(P)H fluorescence are measured. In control samples, a rapid phase of light-driven NADP reduction is overlapped by a somewhat slower phase of NADPH oxidation which is suppressed by iodoacetic acid and, hence, appears to reflect NADPH oxidation by the Calvin cycle. Mercury chloride transforms the light-driven positive response into a negative one, suggesting that inhibition of NADP reduction at the acceptor side of PSI leads to reduction of molecular oxygen, with the hydrogen peroxide formed (via superoxide) causing rapid oxidation of NADPH. The new fluorescence approach opens the way for new insights into the complex interactions between photosynthetic and respiratory pathways in cyanobacteria.     

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria

The induction of a high-affinity state of the CO₂-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO₂ concentrations were resuspended in low-CO₂ buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO₂-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO₂ and HCO₃⁻ fluxes indicated that only the affinity of HCO₃⁻ transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO₂ uptake remained unchanged. Fast induction of high-affinity HCO₃⁻ transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO₃⁻ transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO₃⁻ transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.