Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart.

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).     

Thermodynamic and EPR characteristics of two ferredoxin-type iron-sulfur centers in the succinate-ubiquinone reductase segment of the respiratory chain.

Two distinct ferredosin-type iron-sulfur centers (designated as Centers S-1 and S-2) are present in the soulble succinate dehydrogenase in approximately equivalent concentrations to that of bound flavin. Both Centers S-1 and S-2 exhibit electron paramagnetic resonance absorbance in the reduced state at the same magnetic field (gz = 2.03, gy = 1.93, and gx = 1.91) with similar line shape. Center S-2 is reducible only chemically with dithionite and remains oxidized under physiological conditions. Thus, its functional role is unknown; however, thermodynamic and EPR characterization of this iron-sulfur center has revealed important molecular events related to this dehydrogenase. The midpoint potentials of Centers S-1 and S-2 determined in the soluble succinate dehydrogenase preparations are -5 +/- 15 mV and -400 +/- 15 mV, respectively, while corresponding midpoint potentials determined in particulate preparations, such as succinate-cytochrome c reductase or succinate-ubiquinone reductase, are 0 +/- 15 mV and -260 +/- 15 mV. Reconstitution of soluble succinate dehydrogenase with the cytochrome b-c1 complex is accompanied by a reversion of the Center S-I midpoint from -400 +/- 15 mV to -250 +/- 15 mV with a concomitant restoration of antimycin A-sensitive succinate-cytochrome c reductase activity. There observations indicate that, during the reconstitution process, Center S-I is restored to its original molecular environment. In the reconstitutively active succinate dehydrogenase, the relaxation time of Center S-2 is much shorter than that of S-1, thus Center S-2 spectra are well discernible only below 20 K (at 1 milliwatt of power), while the resonance absorbance of Center S-1 is detectable at higher temperatures and readily saturates below 15 K. Over a wide temperature range the power saturation of Center S-1 resonance absorbance is relieved by Center S-2 in the paramagnetic state, and the Center S-2 central resonance absorbance is broadened by Center S-1 spins, due to a spin-spin interaction between these centers. These observations indicate an adjacent location of these centers in the enzyme molecule. In reconstitutively inactive enzymes, subtle modification of the enzyme structure appears to shift the temperature dependence of Center S-2 relaxation to the higher temperature. Thus the EPR signals of Center S-2 are also detectable at higher temperature. In this system a splitting of the central peak of the Center S-2 spectrum due to spin-spin interaction was observed at extremely low temperatures, while this was not observed in reconstitutively active enzymes or in paritculate preparations. This spin-spin interaction phenomena of inactive enzymes disappeared upon chemical reactivation with concomitant appearance of the reconstitutive activity. These observations provide a close correlation between the molecular integrity of the enzyme and its physiological function.     

Destruction of photosystem I iron-sulfur centers of spinach andAnacystis nidulans by mercurials

Several mercurials destroyed Photosystem I (PSI) Fe−S centers in thylakoids and PSI particles from spinach and fromAnacystis nidulans as revealed by EPR measurement and acid-labile sulfide determination. Of the mercurials tested, HgCl₂ was the most effective, followed by phenylmercuric acetate (PMA), Mersalyl and pCMB in the order of decreasing effectiveness. Fe−S centers in thylakoids were much more labile than those in PSI particles. InA. nidulans thylakoids, Center B was more susceptible than Center A and X to PMA. P700 was less susceptible to PMA than these centers. For 50% inactivation of Fe−S centers inA. nidulans thylakoids, about 0.4 mM PMA was required for Center B, and about 1 mM was required for Center A and X. These differential susceptibilities of Fe−S centers were more pronounced with HgCl₂ than with the other three mercurials.     

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa.

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations. Of the ferredoxin-type iron-sulfure centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two 'high potential iron sulfur' centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished. The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.     

Transmission of bacteriophage T4 amber mutants. I. Temperature sensitivity of gene 26 T4 phage mutant amN131 multiplication in Escherichia coli B cells]

When studying the single cycle of the multiplication of gene 26 mutant amN131 of phage T4, like in temperature shift experiments, the yield of this mutant in non-permissive host depends greatly on the temperature. The burts size of phage in Escherichia coli B is found to be 3.3 phage particles at 25 degrees C, 1.6 at 30 degrees C, 0.051 at 37 degrees C and 0.0007 at 41 degrees C. In the case of permissive host (E. coli CR-63) the burst size per cell decreases from 158 to 49 phage particles at the same temperature interval. The results of the single-burst experiments indicate, that when the incubation temperature increases, the number of E. coli B cells, in which the phage particles maturate, also decreases. It results in the dependence of the transmission coefficient value on the temperature. The transmission coefficient in the conditions favourable for the maturation of the phage is found to be 0.80. It is shown by several methods that the temperature sensitivity of the multiplication of the mutant amN131 in bacterial cells is entirely due to amber mutation in genome of the phage. Therefore the amber mutants having high temperature sensitivity when maturating in non-permissive host cells exist among ordinary amber mutants of phage T4.     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water-splitting activity in photosynthesis.

Plant materials (intact leaves, chloroplasts or subchloroplast particles) pre-illuminated at a low temperature (e.g. -60 degrees C) were rapidly cooled to -196 degrees C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as ZV, A, B1, B2 and C bands. The A, B1, B2 and C bands appeared at constant temperatures, -10, +25, +40 and +55 degrees C, respectively, being independent of the illumination temperature, but the ZV band appeared at a variable temperature slightly higher than the illumination temperature. The B1 and B2 bands were absent in the thermoluminescence profiles of samples devoid of the oxygen-evolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations.

Of the ferredoxin-type iron-sulfur centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two ‘high potential iron sulfur’ centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished.

The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from Center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.

These results are interpreted with respect to the nature and location of the alternative oxidase and with respect to possible models for the nature of the alternative oxygen-consuming component.     

The role of the membrane-bound iron-sulphur centres A and B in the photosystem I reaction centre of spinach chloroplasts.

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I (P-700) and X. Oxidation-reduction potential titrations confirmed that Centre A had Em congruent to -550 mV, Centre B had Em congruent to -585 mV. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, P-700 photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible P-700 photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with Em congruent to -585 mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

Kinetic identification of component X as P430: a primary electron acceptor of Photosystem I

Kinetics of dark recoveries of Component X, Center A, and Center B at 20 and 0 °C after a 30-s illumination were studied in membrane fragments from a blue-green alga by using low temperature electron paramagnetic resonance spectroscopy in combination with a quick-freeze method. These kinetics were compared with those obtained by spectrophotometry under the same conditions. Contrary to the currently popular view, the result strongly suggests that Component X, rather than Center A or Center B, is P430.     

The effect of ambient redox potential on the transient electron spin echo signals observed in chloroplasts and photosynthetic algae

Time-resolved electron spin echo (ESE) studies were carried out at room temperature on chloroplast preparations and whole cells of photosynthetic algae. The signals observed exhibit the unexpected special ESE signal which we have proposed to be the result of transient interactions between P⁺-700 and an early electron acceptor of Photosystem I (Thurnauer, M.C. and Norris, J.R. (1980) Chem. Phys. Lett. 76, 557–561). The intensity of the special ESE signal decreases with the chemical reduction of the Center A-Center B complex. The results suggest that in the untreated photosynthetic systems we are initially observing P⁺-700 as it interacts with the reduced acceptor which precedes the Center A-Center B complex. Then the decay of the special ESE signal (approx. 170 ns) gives the lifetime of this reduced acceptor as it participates in forward electron transport.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

EPR studies by Fe isotopic substitution on the nature of an unknown electron acceptor in Azotobacter vinelandii phosphorylating particles

1. EPR ⁵⁷Fe isotopic substitution studies provide unequivocal evidence that the g = 2.011 signal found in oxidized Azotobacter vinelandii phosphorylating particles is due to an iron-containing structure. The broadening constant determined as a result of this electron—nuclear hyperfine interaction was 15.7 G.

2. A similar signal found in a number of iron—sulfur containing proteins was found by quantitative EPR estimations to exist in a variable but substantial concentration when compared to the intensity of the reduced g = 1.9 type EPR resonance.

3. Reaction of the phosphorylating particles with excess potassium ferricyanide resulted in an alteration of the initial g = 2.011 iron signal resulting in the detection by microwave power studies of at least two different iron species which exhibited major g-values at 1.992 and 2.027.     

Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center 'X' and correlation with optical signal 'A2'

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700+ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center 'X', characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700+ A2 reaction pair and the ESR P-700+ center X- reaction pair to indicate that signal A2 and center X represent the same iron-sulfur center in Photosystem I.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: II. STIMULATION OF THE PASSIVE PERMEABILITY OF CYTOPLASMIC MEMBRANE AT CHILLING TEMPERATURES

Potassium ions and amino acids were found to leak from the cytoplasm to the outer medium when the blue-green alga, Anacystis nidulans, was exposed to the chilling temperatures. The leakage was marked below the critical temperature regions, the midpoint values for which were around 5 and 14 C in cells grown at 28 and 38 C, respectively. These temperature regions coincided with those critical for the susceptibility of the photosynthetic activities and the carotenoid absorption spectrum previously studied (Ono TA, N Murata 1981 Plant Physiol 67: 176-181).     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.     

Transfer of the Excitation Energy in Anacystis nidulans Grown to Obtain Different Pigment Ratios

The blue-green alga, Anacystis nidulans, was grown in lights of different colors and intensities, and its absorption and fluorescence properties were studied. Strong orange light, absorbed mainly by phycocyanin, causes reduction in the ratio of phycocyanin to chlorophyll a; strong red light, absorbed mainly by chlorophyll, causes an increase in this ratio. This confirms the earlier findings of Brody and Emerson (12) on Porphyridum, and of Jones and Myers (8) on Anacystis. Anacystis cultures grown in light of low intensity show, upon excitation of phycocyanin, emission peaks at 600 mmu and 680 mmu, due to the fluorescence of phycocyanin and chlorophyll a, respectively. Changes in the efficiency of energy transfer from phycocyanin to chlorophyll a are revealed by changes in the ratios of these two bands. A decrease in efficiency of energy transfer from phycocyanin to chlorophyll a seems to occur whenever the ratio of chlorophyll a to phycocyanin deviates from the normal. Algae grown in light of high intensity show, upon excitation of phycocyanin, only a fluorescence band at 660 mmu and no band at 680 mmu. This suggests reduced efficiency of energy transfer from phycocyanin to the strongly fluorescent form of chlorophyll a (chlorophyll a(2)) and perhaps increased transfer to the weakly fluorescent form of chlorophyll a (chlorophyll a(1)).     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.