P700 Oxidation Research Articles

CHLOROPHYLL‐PROTEINS AND REACTION CENTER PREPARATIONS FROM PHOTOSYNTHETIC BACTERIA, ALGAE AND HIGHER PLANTS*,‡

Abstract— The use of sodium dodecyl sulfate to dissociate photosynthetic membranes followed by standard fractionation techniques yields chlorophyll‐proteins and reaction center complexes with molecular weights of 500,000 or less. Much about the structure and function of photo‐synthetic units in vivo can be deduced from the properties of the isolated complexes. The Bchl‐protein from green bacteria is approximated by an incompletely filled sphere ˜ 80 Â in diameter consisting of four identical subunits. The five Bchl molecules in each subunit are 14 to 20Â apart. The related Chl a‐proteins from a blue‐green alga and various eukaryotic plants may have similar structural characteristics. The Chl a‐protein from a blue‐green alga contains one molecule of P700 per 60–90 Chl a molecules. The quantum requirement for P700 oxidation is 2.6 or less. The midpoint potential in various preparations ranges from 0.38 V to 0.42 V. Green algae and higher plants yield a Chl a‐protein similar to that from the blue‐green alga; in addition they yield another Chl‐protein (mol. wt. = 2–3×104) which contains an equal amount of Chl a and Chl b. These two Chl‐proteins account for most of the chlorophyll in these organisms. Two photosynthetic bacteria (Rhodopseudomonas viridis and Chromatium) yield protein complexes containing Bchl, carotenoid, and bound cytochromes. The reaction center complex from R. viridis contains P960 (Em, 8= 0.39 to 0.42 V), cytochrome 558 (Em,8= 0.33 V) and cytochrome 553 (Em,7=— 0.02 V). Quantum requirements for P960 and C558 oxidation are ˜2.2 and 3.0, respectively. Complex A from Chromatium contains Bchl 890, P883, cytochrome 556 (Em,8= 0.34 V) and cytochrome 552 (Em,7=˜0.04 V). The quantum requirement for C556 oxidation is about 15. Both high‐ and low‐potential cytochromes can donate electrons to the reaction center chlorophyll present in either complex. This fact supports the idea that only one kind of photochemical reaction center functions in photosynthetic bacteria. An hypothesis about the nature of the photosynthetic unit in purple bacteria is outlined.

P700 oxidation and energy transfer in normal maize and in carotenoid-deficient mutants

1. Particles enriched in System 1 and 2, respectively, were prepared by digitonin fragmentation of chloroplasts from normal maize and from mutants which accumulate lycopene or ζ-carotene instead of the normal carotenoids and which have a reduced chlorophyll content. Although both mutants had a relatively high carotenoid content in relation to chlorophyll, the System-1 particles showed about the same carotenoid to chlorophyll ratio as particles from normal maize. Particles from the lycopene mutant showed approximately a normal ratio of light harvesting to reaction center chlorophyll (P700). In the ζ-carotene mutants the amount of light harvesting chlorophyll was somewhat reduced, both in relation to the number of System-1 reaction centers and to the amount of protein. 2. Action spectra for P700 oxidation indicate a 30–50% efficiency of energy transfer from carotenoids to chlorophyll in System-1 particles of normal maize, increasing with increasing chloroplast development during greening. With the lycopene mutant the transfer efficiency was 30–50%. In the ζ-carotene mutant the transfer efficiency from ζ-carotene was estimated to be somewhat higher. The quantum efficiencies for P700 oxidation for light absorbed by chlorophyll a by System-1 particles from mutant maize were about twice as low as for normal maize. 3. With normal maize, after prior etiolation, the formation of functional System-1 reaction centers lags behind the synthesis of chlorophyll in the early phases of greening. Approximately at the time of beginning of granum formation, when about 20% of the chlorophyll had been synthesized, the ratio of chlorophyll to P700 and the distribution of chlorophyll between the two pigment systems was found to reach a constant value, suggesting a close relation between the regulatory mechanisms for these phenomena and the development of the photosynthetic membrane system.

The reaction between primary and secondary electron acceptors in bacterial photosynthesis

Following a 20-nsec actinic flash, which causes oxidation of P870 and cytochrome C422, Chromatium chromatophores enter a refractory state. While the chromatophores are in this state, a second flash does not cause further oxidation of P870 or cytochrome C422. The quanta of the second flash are wasted as fluorescence (and heat); apparently they do not energize an alternative photochemical reaction. The refractory state probably reflects the accumulation of the primary electron acceptor in a reduced form. By following the reappearance of the capacity for photochemistry, one can measure the kinetics of electron transfer between the primary electron acceptor and the secondary agent which reoxidizes it. In Chromatium chromatophores, this process requires about 60 μsec to proceed half-way to completion at pH 7, and 80 μsec at pH 8. The rate of the reaction increases with decreasing pH, but not in direct proportion to the proton concentration. It increases with temperature, with an E a of about 8.3 kcal/mole. The kinetics are approximately second order in the concentration of the reduced acceptor.

PARTIALLY PURIFIED PHOTOSYNTHETIC REACTION CENTERS FROM PLANT TISSUES

The prior removal of carotenoids from photosynthetic membranes of plants by means of organic extraction allows Triton X-100 to remove and solubilize small particles that are greatly enriched in reaction center Chl, P700. Four plant species have been examined: spinach, bush bean (Phaseolus vulgaris), the blue-green alga Anabaena variabilis, and Euglena. The particles obtained are generally similar and have a ratio of one P700 to approximately 30 chlorophylls (in spinach the ratio is 1 to 28). Exposure of such particles to light or to ferrycyanide causes absorbance changes that are maximal in the vicinity of 700 and 680 nm. The former represents a bleaching (oxidation) of P700, and the latter appears to be caused by a red shift of some Chl closely associated with P700 in the reaction center. The kinetics of P700 oxidation and the fluorescent properties of such preparations indicate a decreased efficiency of energy transfer to the reaction center from the bulk Chl found on the particle.

Kinetic behavior of EPR signal. I. Induction effects in intact algae.

Abstract— There is considerable evidence that the light‐induced narrow EPR signal I at g= 2·0025 in green algae is a reflection of the turnover of the photosynthetic reaction center, P700. A lag period or induction effect of the formation of this signal, subsequent to a dark period in whole fresh cells, is similar to induction effects for the bleaching of P700. The rate of formation of the signal is slower the longer the preceding dark period. DCMU does not much affect the induction phenomenon, but heating the cells to 60° abolishes it. Some substance or process which exists in the intact cell is altered by light and is detectable by its effect on signal I and on the rate of light‐induced oxidation of P700 and cytochrome.

The role of P870 in bacterial photosynthesis

In Chromatium chromatophores, a 30-nsec flash causes an absorbance decrease at 882 nm which is interpreted as an oxidation of P870. The quantum yield is close to one. The reaction is complete in less than 0.5 μsec. It does not occur in the presence of dithionite. With a half-time of 2 μsec, the 882-nm absorbance increases again. Separate measurements show that cytochrome C422 becomes oxidized at the same rate. The 882-nm absorbance increase is interpreted as a reduction of oxidized P870 (P870 +). There appears to be a direct electron transfer between cytochrome C422 and P870 +. The 2-μsec P870 + reduction is generally incomplete at 298 °M. It does not occur at 80 °K, nor does the cytochrome oxidation occur at this temperature. At 298 °K, the addition of N-methylphenazonium methosulfate (PMS) causes the 2-μsec reduction to be complete. In the presence of PMS, the cytochrome oxidation occurs with a quantum yield near one, and its rate and extent are similar to those which occur in whole cells. In Rhodospirillum rubrum chromatophores, the P870 + reduction is much slower. In this species, the initial absorbance changes of P800 are complete within 0.2 μsec. The results support the view that P870 oxidation is the primary chemical reaction of photosynthesis in Chromatium chromatophores.

THE FUNCTION OF P700 AND CYTOCHROME f IN THE PHOTOSYNTHETIC REACTION CENTER OF SYSTEM 1 IN RED ALGAE

Abstract— Kinetics and quantum yields of light‐induced oxidation of P700 and the f‐type cytochrome were measured in marine red algae from absorbance changes in the region 420, 435 and 705 nm. The quantum yield for cytochrome oxidation in Iridaea splendens and Schizymenia pacifica was 0.5‐0.65 in far‐red light, at 20d̀ and at 0d̀C.Oxidation rates of P700 measured when varying amounts of cytochrome were in the oxidized state indicated that a reaction center of system 1 in Iridaea contains 1 P700 and 4 cytochrome molecules. Oxidized P700 only accumulates when all 4 cytochromes are oxidized. The rate of photochemistry of system 1, measured as the sum of the rates of P700 and cytochrome oxidation, was independent of the oxidation level of cytochrome, but decreased with accumulation of oxidized P700. This decrease was less than proportional to the fraction of P700 that was in the oxidized state, which suggested transfer of excitation energy between reaction centers.The quantum yield for cytochrome oxidation after dark periods of 1 min or more was only about 0.2. This effect was tentatively ascribed to a dark reduction of the cytochrome coupled to phosphorylation.