Bacon Ke

Further characterization of a Photosystem-II particle isolated from spinach chloroplasts by triton treatment: The reaction-center components

Abstract Re-examination of the chlorophyll a b ratio in the Photosystem-II reaction-center particle isolated from spinach chloroplasts by Triton treatment yielded a value of 28±2. The Photosystem-II article (as well as the Photosystem-I particle, TSF-1) is enriched in manganese (chlorophyll/Mn, 10 and 10–20, respectively). Heat treatment does not release the manganese from the Photosystem-II reaction-center particle as readily as from native chloroplasts. The fluorescence-induction phenomena of the Photosystem-II particles have been correlated with their photochemical properties in the presence of secondary electron acceptors and (or) donors which further confirm the Photosystem-II character of the particle. C550 and P680, which are currently considered to be the primary electron acceptor and donor, respectively, of Photosystem II, have been detected in the Photosystem-II reaction-center particle by light-induced difference spectra at 77 °K. The presence of P680 has further been corroborated by a free-radical signal revealed by low-temperature EPR spectroscopy. The Photosystem-II particle contains no detectable P700 or bound iron-sulfur proteins. The photobleaching of both C550 and P680 is practically irreversible at 77 °K. The Photosystem-II reaction-center particle is enriched in P680, C550 as well as cytochrome b 559 approximately 10-fold relative to the unfractionated chloroplasts.

Protochlorophyllide aggregation in solution and associated spectral changes

Abstract When protochlorophyllide was dissolved in solvents of low dielectric constants ( e.g. , benzene, chloroform, diethyl ether, etc. ), its absorption spectrum showed a red shift with time. For instance, in 5 h, the original absorption bands in benzene at 634, 581 and 539 nm were shifted to 651, 587 and 546 nm. In addition, the blue band at 443 nm was split into two bands at 442 and 472 nm. The red shift was reversed upon addition of a trace amount of a more polar solvent such as methanol and upon standing. Also, in more polar solvents, no red shift in the absorption spectrum occurred. The time-dependent spectrum shift was accompanied by a loss of fluorescence intensity at 640 nm and by an increase in scattering of the 440 nm excitation light. Again, these changes could be reversed by a trace amount of a more polar solvent. The red shift in the absorption spectrum, the decrease in fluorescence intensity and the increase in light scattering were interpreted as due to an aggregation of the pigment molecules.

Optical rotatory dispersion of chloroplast-lamellae fragments

Abstract The optical rotatory dispersion (ORD) of the chloroplast-lamellae fragments shows Cotton effects corresponding to all the pigment-absorption peaks in the visible region and a negative trough at 235 mμ. The ORD spectrum in the visible region shows considerably more complexity and intensity than that of the isolated chlorophylls or xanthophylls in an organic solvent. The enhancement of the intrinsic Cotton effects of the pigments in the lipoprotein suggests a strong interaction between the pigment molecules themselves and/or between the pigment molecules and the attached lipoprotein macromolecules. The major contribution to the 235 mμ trough was attributed to protein conformation. Under this assumption, the protein in the chloroplst lamellae was estimated to contain approximately 17% helix. Heating the chloroplast lamellae reduced the magnitude of the 235 mμ trough and shifted the characteristic ORD spectrum of the native lamellae to one resembling that of the free pigments. On the other hand, urea treatment of the lamellae protein only reduced the 235 mμ trough but did not affect the visible-region Cotton effects. Heating the lamellae possibly caused a disruption of the organizational relationship between the pigment and the lipoprotein as well as the helical conformation.

Experimental determination of the molar differential extinction coefficient of P700

Abstract The molar differential extinction coefficients of P700 at 435 and 703 nm have been determined by using a directly coupled reaction in the dark involving the reduction of photooxidized P700 and the oxidation of reduced cytochromes. From the coupled oxidation of mammalian cytochrome c by Anabaena high-P700 particles, the molar differential extinction coefficients of P700 at 435 and 703 nm are calculated to be 8.6·104 and 1.20·105 M−1·cm−1, respectively, and the ratio of the red to blue band heights is 1.4. From the coupled oxidation of Euglena cytochrome-552 by Triton-fractionated pigment system 1 subchloroplast particles enriched in P700, the calculated molar differential extinction coefficients of P700 at 435 and 703 nm are very close to the values given above. Using an absorbance decrease at 580 nm as a measure of the photoreduction of dichlorophenolindophenol by Triton-fractionated pigment system 1 subchloroplast particles enriched in P700 tends to yield a low extinction value because of other absorbance changes which occur at this wavelength and the non-reproducibility of the values obtained. Comparisons are made between the extinction values of P700 and the corresponding extinction values of the bacteriochlorophyll reaction centers in photosynthetic bacteria.

LIGHT-INDUCED RAPID ABSORPTION CHANGES DURING PHOTOSYNTHESIS. V. DIGITONIN-TREATED CHLOROPLASTS.

Abstract Fresh chloroplasts treated with appropriate amounts of digitonin give light-induced absorption changes in the Soret-band and far-red regions. The absorption changes are enhanced by ascorbate and eliminated by ferricyanide. The light-minus-dark difference spectrum of digitonin-treated chloroplasts agrees closely with that of the pigment complex P 700 reported by Kok . Dichlorophenolindophenol does not appear to mediate in the electron transfer between ascorbate and the photooxidized chlorophyll complex, whereas phenazine methosulfate, depending on its concentration, greately modifies the kinetics of the re-duction of the pigment complex by ascorbate.

Light-induced rapid absorption changes during photosynthesis. VIII. Cytochrome and bacteriochlorophyll reactions in Rhodospirillum rubrum cells

Abstract By means of flash spectrophotometry, the oxidation reactions of cytochromes c 2 and b can be differentiated. Cytochrome c 2 oxidation had a risetime of 1–2 msec; that of cytochrome b was several times longer. Both risetimes were several orders of magnitude longer than the duration of the excitation flash, suggesting that both cytochromes were oxidized in the dark. Unique for the cytochrome- b transients was the appearance of sharp spikes occurring always in the direction opposite of those of the cytochrome- b oxidation. These transient spikes occurred at an excitation intensity level beyond the saturation level for both cytochromes c 2 and b . The risetime for the sharp spike transients was approx. 50 μsec. In the wild-type Rhodospirillum rubrum cells, reactions due to the carotenoids caused spectral changes in the 470–560-mμ region. This resulted in an unproportionately large negative α- and β-bands in the light- minus -dark difference spectrum. There was no noticeable difference in the decay kinetics between the cytochromes and the carotenoids. The oxidation of both cytochromes c 2 and b was stopped near o°. At −196°, positive bands appeared at 430 and 790 mμ, and negative bands appeared at 380, 810 and 870 mμ. All reactions taking place at the liquid-nitrogen temperature had a risetime of approx. 50 μsec. The low-temperature difference spectrum probably represents changes originated from the bacteriochlorophyll molecules. The net effect of 4 inhibitors ( p -chloromercuribenzoate (PCMB), phenylmercuriacetate, 2- n -heptyl-4-hydroxyquinoline- N -oxide (HOQNO), and antimycin A) on R. rubrum cells was similar, i.e. , the light- minus -dark difference spectrum resembled closely that obtained with whole cells at −196° or from chromatophores at room temperature. The effect of PCMB on the transients at all wavelengths was immediate. In the presence of phenylmercuriacetate, HOQNO or antimycin A, however, the final transient profile at 420 mμ took more than 2 h to become established. Prior to that time, the transient underwent a series of intermediate stages. More unusally, the 420-mμ transient in the presence of HOQNO or antFSimycin A appeared to completely revert to the initial profile at approx. 120 min after the inhibitor was added.

CHLOROPHYLL MONOLAYERS AND MUTILAYERS—I. METHODS OF PREPARATION AND SPECTROSCOPIC CHARACTERIZATION*

Abstract— Precise methods of preparing monolayers and multilayers of chlorophyll a alone or mixed with arachidic acid on glass slides prewated with monolayers of barium arachidate are described in detail. Chlorophyll monolayers obtained by this method are mechanically stable and allow the absorption spectra or other spectroscopic characterizations to be readily made.

Light-induced rapid absorption changes during photosynthesis. VI. Complex reactions in some blue-green algae.

Abstract Depending on the measuring wavelengths, light-induced complex absorption-change transients with single exponential or biphasic decay have been observed in a number of blue-green algae. Detailed study with Plectonema boryanum revealed four types of transients with characteristic rise and decay kinetics: 1. (1) The rapidly rising (rise-time ⩽ 50 μ sec) and slowly decaying (half-time 300 ± 50 msec) transients, or S-transients. 2. (2) The rapidly rising and rapidly decaying (half-time 7 msec) transients, or R-transients. 3. (3) The rapidly rising and rapidly decaying (half-time 1.5 msec) transients. 4. (4) The slowly rising (∼ 10 msec) and slowly decaying (150 ± 50 msec) transients. In fresh blue-green cells, the S-(type 1) and R-transients (type 2) are always observed and reproducible, and the ratio R/S is near unity. In some blue-green algae, the R-transients deteriorate upon storage. The R-transients appear only when the excitation-beam intensity exceeds a certain threshold, but is independent of the measuring-beam intensity. Transients of types (3) and (4) are observed only part of the time. The light- minus -dark difference spectrum suggests that the rapidly rising and slowly decaying S-transients (type 1) represent the photooxidation of a cytochrome and the chlorophyll complex P 700 located at or near pigment system 1. However, there is no apparent evidence of a sequential reaction among these two species. The chemical nature of the other three types of transients remains unclear at this time. Heating up to 50° for 5 min has no effect on the 430-mμ biphasic transient. Heating near 60° preferentially affects the R-portion of the biphasic transient. Above 62°, the S-portion is also largely eliminated. Sonicated cell fragments yield a composite signal almost identical to that of fresh cells, except the overall magnitude is slightly reduced. The signal is stable between pH 4 and 11.8. 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) has little effect on the composite signal at 430 mμ, except the R/S ratio becomes slightly smaller, and the decay of the S-transient becomes slower. Parallel experiments on heating, sonication or DCMU treatment with oxygen evolution yield no correlation between the latter and the complex absorption-change transient. Phenylmercuric acetate at 10 −4 M shifts the oxidation level of cytochrome and P 700 in pigment system 1, and subsequent illumination causes their reduction, as manifested in the reversal of sign of the S-portion of the composite transients at 430 and 700 mμ. The simultaneous presence of phenylmercuric acetate and DCMU eliminates the composite signal completely. Addition of 10 −2 M ascorbate has no effect in relieving the inhibition, but ascorbate plus trace amount of dichlorophenol-indophenol or di(or tetra)methyl- n -phenylene diamine restored the complete biphasic signal. The effectiveness spectra obtained with 10-mμ-wide flashes indicate that 680-mμ light is active in producing the S-transients, whereas the R-transients can be produced by far-red light absorbed by chlorophyll as well as by 620-mμ light absorbed by phycocyanin. Interaction between the two pigment systems is demonstrated by the acceleration of decay of the S-transient when 680- and 620-mμ flashes are applied simultaneously, and by the absence of such an acceleration when different junctions of the electron-transport chain are blocked by DCMU or 2- n -heptyl-hydroxyquinoline- N -oxide. Interaction between the two pigment systems is further demonstrated by using one light for steady background illumination and another light for flash excitation. For instance, under steady illumination with 680-mμ light, a 620-mμ flash reverses the sign of the S-transient, indicating that 680-mμ light generates oxidized cytochrome and P 700 from pigment system 1 and subsequent 620-mμ flash generates a reductant from pigment system 2.

Light-induced rapid absorption changes during photosynthesis: IV. Reactions in aged chloroplasts in the presence of ascorbate and redox dyes☆

Abstract Transient absorption changes at 430 mμ are induced in aged chloroplasts by red light flashes, The absorption change occurs in 10 −4 sec or less and has a half life of approx. 10 −2 sec. The enhancement of the reaction by ascorbate and the abolishment by ferricyanide suggest that an oxidation reaction is responsible for the absorption change. In the presence of 3-(3,4-dichlorophenyl)- I,I -dimethylurea the absorption change is not observed, but the signal can be restored completely by adding ascrobate. Available evidence from the decay kinetics of the 430-mμ transient absorption changes in the presence of ascorbate suggests that dichlorophenolindophenol at all concentrations reacts with cytochrome while phenazine methosulfate at low concentrations reacts with cytochrome and at concentrations greater than 3.10 −5 M with P700 only. This in accord with the difference spectra observed in the blue region (where also cytochrome changes are observed) and also with the concominant and similarly decaying absorption changes at 430 and 703 mμ. Light-intensity dependency of the complex reaction in aged chloroplast containing ascorbate and trace amount of phenazine methosulfate showed that at low intensity only the oxidation of the chlorophyll complex takes place. The coupling reaction between the pigment complex and cytochrome takes place only at higher light intensities.

CHLOROPHYLL MONOLAYERS AND MULTLLAYERS—II. EVIDENCE FOR THE PRESENCE OF ORDERED AGGREGATES*

Abstract— Results from measurements of the absorption spectrum and dichroism suggest that oriented chlorophyll aggregates can be prepared by the monolayer techniques described in the preceding paper.(1)