Warren L. Butler

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone

The quenching action of dibromothymoquinone on fluorescence and on primary photochemistry was examined in chloroplasts at minus 196 degrees C. Both the initial (F0) and final (FM) levels of fluorescence as well as the fluorescence of variable yield (FV equals FM minus FO) were quenched at minus 196 degrees C to a degree which depended on the concentration of dibromothymoquinone added prior to freezing. The initial rate of photoreduction of C-550 at minus 196 degrees C, which was assumed to be proportional to maximum yield for primary photochemistry, phipo, was also decreased in the presence of dibromothymoquinone. Simple theory predicts that the ratio FV/FM should equal phipo. Excellent agreement was found in a comparison of relative values of phipo with relative values of FV/FM at various degrees of quenching by dibromothymoquinone. These results are taken to indicate that FO and FV are the same type of fluorescence, both emanating from the bulk chlorophyll of Photosystem II. Dibromothymoquinone appears to create quenching centers in the bulk chlorophyll of Photosystem II which compete with the reaction centers for excitation energy. The rate constant for the quenching of excitation energy by dibromothymoquinone is directly proportional to the concentration of the quencher. Rate constants for the de-excitation of excited chlorophyll molecules by fluorescence, kF, by nonradiative decay processes, kD, by photochemistry, kP, and by the specific quenching of dibromothymoquinone, kQ, were calculated assuming the absolute yield of fluorescence at FO to be either 0.02 or 0.05.

Fluorescence quenching in Photosystem II of chloroplasts

A simple photochemical model for the photosynthetic units of Photosystem II based on first-order rate constants for de-excitation of excited chlorophyll molecules is presented in the form of equations which predict the yields of fluorescence (i.e. at the FO level, at the maximal FM level and the fluorescence of variable yield, FV equals FM minus FO). Two types of quenching mechanisms are recognized: (1) increasing nonradiative decay processes in the bulk chlorophyll by creating quenching centers which complete with the reaction centers for the excitation energy (this mechanism quenches both FO and FV) and (2) increasing nonradiative decay of the excited reaction center chlorophyll (this mechanism quenches FV but not FO). Quenching in the bulk chlorophyll preserves the relationship that Fv/FM is equal to the maximum yield of photochemistry; quenching at the reaction center chlorophyll decreases FV/FM substantially (since FV is quenched specifically) but may have very little effect on the yield of photochemistry. Estimates are made of the relative magnitudes of the rate constants for de-excitation of the excited reaction center chlorophyll by photochemistry, kp, by nonradiative decay processes, kd, and by energy transfer back to the bulk chlorophyll, kt. Fluorescence is assumed to emanate only from the bulk chlorophyll. Energy transfer from Photosystem II to Photosystem I may occur from either the excited bulk chlorophyll or from the excited reaction center chlorophyll. The model is valid for any degree of energy transfer between Photosystem II units.

HIGHER DERIVATIVE ANALYSIS OF COMPLEX ABSORPTION SPECTRA

Abstract— –A small computer on line with a single‐beam spectrophotometer was used to obtain absorption spectra and higher derivatives (up to the fourth) of the absorption spectra. The spectral resolution was markedly enhanced in the higher derivative curves. The computer was also used to synthesize absorption spectra from Gaussian, Lorentzian and mixed Gaussian‐Lorentzian band shapes in order to explore the higher derivative analysis of completely defined spectra.

Energy transfer between Photosystem II and Photosystem I in chloroplasts

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at - 196 degrees C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5mMMgCl2 which presumably changes the distrubution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, alpha, being the fraction distributed to Photosystem I, and beta, the fraction to Photosystem II to Photosystem I, KT(II yields I). The data, analyzed within the context of the model, permit a direct comparison of alpha and kt(II yields I) in the absence (minus) and presence (+) of Mg-2+ :alpha minus/alpha-+ equals 1.2 and k-minus t)II yields I)/K-+T(II yields I) equal to 1.9. If the criterion that alpha + beta equal to 1 is applied absolute values can be calculated: in the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg-2+, alpha-minus equal to 0.32 and phi t(II yields I) varied from 0.12 to 0.28. The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvesting chlorophyll of Photosystem II to Photosystem I, kt(II yields I), and a transfer from the reaction centers of Photosystem II to Photosystem I, kt(II yields I). In that case alpha-minus/alpha+ equal to 1.3, k-minus t(II yields I)/k+ t(II yields I)equal to 1.3 and k-minus t(II yields I) equal to 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.

ACTTON SPECTRA OF PHYTOCHROME IN VITRO

Summary First‐order rate constants, Kλ, for the photochemical conversions of both forms of phytochrome, PR and PFR, were measured at various wavelengths between 300 and 800 nm. The product of the extinction coefficient, ε, and the quantum yield, ø, at any wave‐length, λ, could be determined from the value of Kλ and the mole fractions of PR and PFR present at the photostationary state set by λ. The determination of the action (εø) spectra required that the photostationary states in red and far‐red light be known. These were determined from the absorption spectra and kinetic data.

Absorption of Light by Turbid Materials

The transmission and absorption properties of turbid media have been examined with Kubelka and Munk’s theory of the optics of intensely scattering material. The equation for the optical density of such material as a function of thickness has been derived and examined experimentally. It is shown that the reflectivity and scattering coefficient can be determined absolutely without reference to a standard material from the optical-density measurements. The absorption spectra of pigments in scattering media and in clear solution have been compared. It is shown that light in passing through a turbid sample may traverse an optical path which is many times the sample thickness. The practical consequence of this increased path length is an intensification of the absorption bands of pigments in light-scattering media. The theoretical expression for this intensification has been derived and tested experimentally. Spectral effects due to the physical binding of pigment molecules to the scattering particles have also been examined.

The relationship between Q, C-550 and cytochrome b 559 in photoreactions at −196° in chloroplasts

Abstract Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.

AN ANALYSIS OF FOURTH DERIVATIVE SPECTRA

Abstract— –Fourth derivative spectra of chloroplast at – 196°C, obtained by four sequential differentiations of the absorption spectrum, were examined to determine the conditions for optimal signal‐to‐noise ratio and resolution. An appreciable improvement in the signal‐to‐noise ratio was found when the four differentiating intervals were nearly but not exactly equal. The process of differentiating digitized data with the computer was explored in order to define the rules for optimization of the fourth derivative curves.

Potentiometric titration of the fluorescence yield of spinach chloroplasts

Abstract The fluorescence yield of spinach chloroplasts was measured as a function of oxidation-reduction potential under anaerobic conditions at pH 6.0, 7.0, and 8.0. The potentiometric-titration curve showed two fluorescence quenching processes, both quenching in the oxidized state. The complete titration curve could be reasonably fit with a Nernst equation for the sum of two one-electron-transfer reactions with different midpoint potentials. At pH 7.0 the two transitions had midpoint potentials of about −35 mV and −270 mV and both showed a pH dependence of about −60 mV per pH unit. The hypothetical quencher, Q, assumed to be responsible for light-induced fluorescence-yield changes in green photosynthetic systems could be ascribed to the more positive quenching component.

The spectrophotometry of dense light-scattering material.

Abstract The spectrophotometry of dense light-scattering samples by means of a single-beam recording spectrophotometer is described. The optical path length of light traversing a light-scattering sample may be many times the sample depth. By adding CaCO 3 to reduced cytochrome c solutions, the intensity of absorption bands was intensified 70-fold as the result of a 70-fold increase of path length. A similar intensification of over tenfold was shown to exist in apple tissue. The photoreversible pigment which controls so many developmental responses of plants is demonstrated spectrophotometrically in intact corn coleoptiles. The spectra of dry yeast as well as dense yeast suspensions are presented. In addition to the typical cytochrome complement, a cyanide-sensitive absorption band at 640 mμ is shown. The spectra of intact lima bean seeds, both dry and imbibed, show spectral changes associated with germination processes.