David Mauzerall

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients. Two independent sets of measurements using a variety of methods show that a sample with A800 nm = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1. Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77. These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: e802 nm = 288 (± 14) mM−1 · cm−1 and e865 nm = 128 (± 6) mM−1 · cm−1. For reversible bleaching of the 865-nm band, Δered - ox865nm = 112 (± 6) mM−1 · cm−1 (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.

Absolute absorption cross-sections for Photosystem II and the minimum quantum requirement for photosynthesis in Chlorella vulgaris

Abstract Absolute absorption cross-sections for oxygen production (σO2) were determined from the light-saturation behavior of oxygen flash yields from whole cells of Chlorella vulgaris illuminated with submicrosecond flashes of laser light. Light-saturation curves were well described by simple Poisson statistics with a single average cross-section per photosystem trap (RCII). The maximum variation about the average cross-section permitted by the data was a factor of 3. σO2 at the laser wavelength (596 nm) increased from 38 A2 for cells grown in high light to 115 A2 for cells grown in low light. The 3-fold variation in σO2 was accompanied by a 10-fold variation in total cell chlorophyll content. This behavior results, at least in part, from the partitioning of chlorophyll between Photosystem II (measured) and Photosystem I (unmeasured). The 596 nm in vivo absorption cross-section for chlorophyll in Chlorella (σChl) is 0.29 A2, independent of total cell chlorophyll content. The antenna size of RCII was calculated to range from 130 to 400 molecules of chlorophyll. At low flash energies, the relationship between the quantum requirement for oxygen production (QR), the maximum oxygen-flash yield or Emerson and Arnold number (PSUO2) and our cross-sections is QR =( PSU O 2 )· ( (σ Chl ) (σ O 2 ) . QR, found to be independent of both total cell pigmentation and RCII antenna size, was constant at 10±1 photons absorbed per oxygen molecule evolved.

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae.

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.

Relationship of steady-state photosynthesis to fluorescence in eucaryotic algae

Abstract The change in fluorescence yield (Δφ) was measured in five species of eucaryotic algae using a ‘pump and probe’ flash technique. The half-time for the oxidation of Q, which was measured by varying the delay time between actinic (pump) and measuring (probe) flashes, averaged 400 μs and was unaffected by background irradiance between 10 12 and 10 16 quanta · cm −1 · s −1 . The absorption cross-section of PS II traps was measured by varying the intensity of the actinic flash. These cross-sections did not change (± 10%) with background irradiance. The cross-section data can be fitted to a cumulative one-hit Poisson distribution. In the steady state, the relationship between δφ and photosynthetic oxygen evolution was highly nonlinear and cannot be explained by energy transfer between PS II units. Using the criteria of Δφ, about 15% of the PS II traps remain open at light saturation of O 2 evolution. Conversely, at low irradiance levels, capable of stimulating much less than 1% of the maximum steady-state photosynthetic rate, the fluorescence yield decreases by as much as 25% from the dark-adapted value. Furthermore, the data suggest that a long-lived quencher of fluorescence is formed at moderate to continuous irradiance levels, at least 10 16 quanta · cm −2 · s −1 . Our results suggest that cyclic electron flow around PS II occurs under normal physiological conditions and is especially pronounced in chlorophytes.

The absolute size of a photosynthetic unit

Summary The photosynthetic unit is a complex of pigments and proteins coupled to a reaction center where the initial light-driven charge separation of photosynthetic reactions takes place. The ‘size’ of the photosynthetic unit is defined as the number of light-absorbing pigment molecules contributing excitation energy to a reaction center. It is an important parameter in the study of the structure and function of photosynthetic membranes. Several approaches used for relative and absolute measures of photosystem stoichiometry and size are evaluated, and results achieved by these methods are compared. The concept of the absolute size of a photosynthetic unit leads to a simple relation between the size of the oxygen forming unit, the total chlorophyll per O2 (the classical Emerson-Arnold unit), and the quantum requirement for O2 formation. These measurements lead to further understanding of the mechanisms of energy transfer and of the adaptive mechanisms of plants to varied environments.

Characterization of primary reactants in bacterial photosynthesis. I. Comparison of the light-induced EPR signal (g=2.0026) with that of a bacteriochlorophyll radical.

Abstract We show in this work that the light-induced free radical in Rhodospirillum rubrum arises from a radical of bacteriochlorophyll. The identification is based on the comparison of the EPR characteristics ( g values and line widths) of the light-induced radical with those of a bacteriochlorophyll radical prepared in vitro . The g values are found to be identical ( g = 2.0026 ± 0.0001) within the experimental error. Comparison of the linewidth components of native and deuterated radicals at two microwave frequencies shows identity of several aspects of the electronic structure of the two radical species. The similarity of the line widths of the EPR signals in different bacterial species suggests that these conclusions can be generalized to the other photosynthetic bacteria.

Effects of Iron Limitation on Photosystem II Composition and Light Utilization in Dunaliella tertiolecta

The effects of iron limitation on photosystem II (PSII) composition and photochemical energy conversion efficiency were studied in the unicellular chlorophyte alga Dunaliella tertiolecta. The quantum yield of photochemistry in PSII, inferred from changes in variable fluorescence normalized to the maximum fluorescence yield, was markedly lower in iron-limited cells and increased 3-fold within 20 h following the addition of iron. The decrease in the quantum yield of photochemistry was correlated with increased fluorescence emission from the antenna. In iron-limited cells, flash intensity saturation profiles of variable fluorescence closely followed a cumulative one-hit Poisson model, suggesting that PSII reaction centers are energetically isolated, whereas in iron-replete cells, the slope of the profile was steeper and the calculated probability of energy transfer between reaction centers increased to >0.6. Immunoassays revealed that in iron-limited cells the reaction center proteins, D1, CP43, and CP47, were markedly reduced relative to the peripheral light-harvesting Chl-protein complex of PSII, whereas the [alpha] subunit of cytochrome b559 was about 10-fold higher. Spectroscopic analysis established that the cytochrome b559 peptide did not contain an associated functional heme. We conclude that the photochemical conversion of absorbed excitation energy in iron-limited cells is limited by the number of photochemical traps per unit antenna.

Feedback controlling oxygen production in a cross-reaction between two photosystems in photosynthesis

Abstract The non-linear light-saturation curve for oxygen production in both Chlorella vulgaris and Phormidium luridium at low light intensities, under anaerobic conditions is shown to be caused by the reduction of a pool of electron carriers coupled to both an endogenous reducing agent R, and to oxygen. The light dependence of oxygen production in these algae was studied by a repetitive-flash method, which allows a direct analysis of the steady-state kinetics of pool reduction. We propose a kinetic model which quantitatively accounts for these kinetics and several transient phenomena. This model centers on a novel cross reaction at the pool of photo and dark electron input and output, allowing a delicate poising of oxygen production by the environment. This model shows a positive feedback of oxygen on oxygen production.

Calcium and photosynthetic oxygen evolution in cyanobacteria

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca(2+). Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca(2+) addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca(2+) and Mg(2+) stimulate turnover, unit activation is Ca(2+) specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca(2+) -activated preparation. The Ca(2+) requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca(2+) of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605--609) these observations strongly suggest a site of Ca(2+) action within Photosystem II. The pronounced specificity of the Ca(2+) requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca(2+) dependence in P. luridum, French-press treatment is required for maximal expression of Ca(2+) -specific effects. French-press breakage causes a release of endogenous Ca(2+) from cells, supporting the view that added Ca(2+) restores oxygen evolution by satisfying a physiological requirement for the cation.

THE PHOTOOXYGENATION OF MAGNESIUM‐OCTAETHYLPORPHIN*

Abstract— Octaethylporphinato‐magnesium (MgOEP) has been photooxygenated in quantitative yield to a brown compound with its main absorption peaks at 825 and 408 nm. The product has been isolated and evidence suggests that it is 4‐formyl‐5‐oxa‐octaethylporphinato‐magnesium II. II is very labile to hydrolysis, the main isolated decomposition product being 1, γ‐dioxo‐8‘‐formyl‐octaethyl violin III. The absorption, i.r., PMR and mass spectra of II and III are reported and possible paths to their formation are discussed.