Studies on the cytochromef ofBryopsis maxima in vivo andin vitro

Cytochromec (553.7Bryopsis maxima) isolated fromB. maxima had absorption maxima at 553.7, 523.0, 417.1 and 317.5 nm in its reduced form. Isosbestic points in the reduced minus oxidized difference spectrum were located at 561, 543, 528, 511, 436, 411 and 334 nm. The purified protein exhibited a molecular weight of 10,700. The midpoint potential for the cytochromec was estimated to be 372±5 mVin vitro at pH 7.0 and 365±5 mVin vivo.In vivo 80% of the cytochromec was in the reduced form. This cytochrome was located only in chloroplasts indicating that it functions in the photosynthetic electron transport as cytochromef. Chloroplasts contained one molecule of this cytochrome per 360 molecules of chlorophyll. The magnitude of the chemically induced absorbance changes for the cytochromoesin vivo were much smaller than the light-induced absorbance change at 561 nm. It is concluded that the light-induced 561 nm absorbance change characteristic of this alga is not mainly attributable to the redox reaction of cytochromesb andf in the chloroplasts.     

Changes in Pigment Composition and Photosynthesis Induced by Iron-Deficiency in the Blue-Green Alga Anacystis nidulans

Absorption spectra and photosynthetic action spectra have been determined for living Anacystis grown in complete and iron-deficient inorganic media. The absorption studies have shown a spectral shift from 679 nm to 673 nm in the chlorophyll a absorption peak when the algae had to grow without iron. The shift is believed to reflect a changed ratio between at least two chlorophyll a forms denoted Ca670 and Ca680 in this work. Action spectra determinations have revealed a similar shift from 677 nm to 672 nm in the photosynthetic activity peak of chlorophyll a when Anacystis was transferred to a medium without iron. It is proposed that both Ca670 and Ca680 participate in light absorption for photo-system I.     

Action Spectra for the Transient and the Normal Photosynthetic Oxygen Evolution in Wheat Leaves

An action spectrum within the wavelength region 430–700 nm has been determined for an oxygen Iransient isolated from the main photosynthetic oxygen evolution in wheat leaves. the spectrum follows the absorption spectrum of chlorophyll b. In order to compare this oxygen evolving process with the normal photosynthetic oxygen evolution, an action spectrum for this later process was also determined but only in the wavelength region 600–700 nm. This action spectrum closely corresponds to other action spectra for the photosynthetic oxygen evolution found in the literature. The behaviour of the oxygen transient after various treatments was also studied. It was found that the transient oxygen evolution was influenced by the dark period between irradiations, and by a previous irradiation with a wavelength longer than 695 nm, both conditions having a promoting effect on the oxygen evolution. The action spectra and the other findings are discussed on the basis of the two-pigment hypothesis.     

Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature.

We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.     

Action Spectra of Photosynthetic Activities of Chlorella ellipsoidea

• 1) Suspensions of Chlorella show an even stronger light scattering than suspensions of chloroplasts of spinach. The bands of absorption are thus broadened and, at higher concentrations, moved to lower wave-lengths. The intensity of the photosynthesis closely follows the curves of light scattering, a fact partly explaining the high efficiency of green light. Calculated per unit thermoelectrically measured incident energy the action spectrum shows bands at 660–670 nm and c. 500 nm and a comparatively high level of the whole region 500–560 nm.
• 2) Flash experiments show the existence of a steady state carotene/xanthophyll that is moved to reduction (c/x > 1) in blue and green light and to oxidation (c/x < 1) in red light. All experiments point to the existence of two light reactions, the first one involving excitation of carotenoids, with ferredoxin-TPN as acceptor, the second one involving excitation of chlorophyll, with the cytochrome system of the chloroplasts acting as donors of electrons and thus completing an energy converting circulation between pigments and enzyme systems.
• 3) The operation of combined light reactions appears also from experiments with simultaneous or succedaneous illumination with monochromatic light of different wave-lengths. Some effects may be explained from separate excitations of carotenoids and chlorophylls, others may depend on still unknown photic reactions.
• 4) The action spectrum in ultrared shows a positive band at c. 900 nm but no or only very small effects in the region 950–1400 nm. Ultrared radiation has on the other hand an enhancing effect on the light excitation in the visible spectrum. A combination of infrared and visible radiation shows a roughly linear relation between incident energy and photosynthetic effect.
• 5) All experiments were performed in the region of linear relation between intensity of incident light and O₂-production. Induced effects of combined monochromatic regions show a very rapid initial change in the steady states that in one or two minutes simmers down to a balanced state of continued photosynthesis. No change was observed in the total quantity of the pigments.

     

Purification and some properties of a soluble cytochrome (cytochrome c-551) from Erythrobacter species strain OCh 114

A soluble cytochrome, cytochrome c-551 was purified from an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942) by ammonium sulfate fractionation, ion-exchange chromatography and gel-filtration. The cytochrome had absorption maxima at 277, 410, and 524–525 nm in the oxidized form, and at 415, 522, and 550.5 nm in the reduced form. At 77 K, the -band of the absorption spectrum of the reduced form split in two at 547 and 549 nm. The millimolar absorption coefficient at 550.5 nm was 26.8 mM^-1 cm^-1 in the reduced form. This cytochrome was an acidic protein with an isoelectric point of 4.9. Its molecular weight was determined to be 15,000 by gel-filtration on Sephadex G-100 and 14,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The midpoint potential of this cytochrome was +250 mV at pH 7.0. This cytochrome did not bind CO.     

Iron Deficiency in the Blue-Green Alga Anacystis nidulans: Changes in Pigmentation and Photosynthesis

Phycocyanin-free photosynthetic lamellae (PSI-particles) were prepared from Anacystis nidulans, grown in complete and iron-deficient media. French press treatment and fractionated centrifugation were used. Absorption studies of the particles revealed an iron deficiency-induced shift of the main red chlorophyll a absorption peak from 679 to 673 nm as reported before for whole cells. The shift may reflect a changed distribution between different chlorophyll a forms. Action spectra for photo-oxidation of mammalian cytochrome c with photosynthetic lamellae revealed an iron deficiency-induced shift, corresponding to that found in the absorption spectra. As photo-oxidation of cytochrome c is mediated by PSI, it is believed that chlorophyll a also after the shift towards shorter wavelengths, is active in PSI. A decreased photosynthetic capacity of PSI, due to iron deficiency, was shown by time course studies of photosynthetic oxygen evolution, by photo-oxidation studies of P700 and mammalian cytochrome c, by photo-reduction studies of NADP and by combined studies of light-induced and chemical oxidation of P700. The ration chlorophyll a/700 was also determined for whole cells, lyophilized cells and PSI-particles. Iron deficiency caused an increased ratio in all studied fractions. The results of this work imply that energy is transferred with less efficiency within the photosynthetic units of PSI in iron-deficient A. nidulans than in iron-supplied algae.     

Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Ascorbate-independent carotenoid de-epoxidation in intact spinach chloroplasts

Slow (> 1 s) light-induced absorbance changes in the 475–530 nm spectral region were examined in Type A chloroplasts from spinach. The most prominent absorption change occurred at 505 nm. The difference spectrum for this light-induced increase, its absence in osmotically shocked chloroplasts and restoration by ascorbate, and its sensitivity to dithiothreitol indicate that the absorption change is due to carotenoid de-epoxidation. The reaction in intact chloroplasts is characterized by its independence of exogenous ascorbate and a rate constant 3- to 8-fold higher than that reported previously for chloroplasts supplemented with ascorbate.The relevance of carotenoid de-epoxidation to other photosynthetic processes was examined by comparing their sensitivities to dithiothreitol. Levels of dithiothreitol that eliminate the 505 nm shift are without effect on saturated rates of CO₂ fixation and do not appreciably inhibit fluorescence quenching. We conclude that carotenoid de-epoxidation is not directly involved in the reactions of photosynthesis or in the regulation of excitation allocation between the photosystems.     

Binding of ligands and spectral shifts in cytochrome c oxidase

1. On addition of reductant (ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa(3)), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415nm, 445nm and 605nm, which slowly gives rise to the final ;half-reduced' species in whose spectrum the 415nm peak has disappeared and a new absorption is seen at 430-435nm. 2. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the ;low-spin' cyanide form with a peak at 432nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally ;mixed-spin', undergoes no further changes. 3. The Soret-peak shift of oxidized cytochrome a(3) which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605nm to 603nm. A partial blue shift of the alpha-peak occurs in the half-reduced sulphide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulphides [a(2+)a(3) (3+)HSR compounds, where R=CH(3), C(2)H(5) or (CH(3))(2)CH]. 4. Analogous, albeit less readily decipherable, spectroscopic effects with the ligands imidazole and alkyl isocyanides suggest that on reduction of cytochrome a an interaction occurs between the two haem groups involving (i) a high- to low-spin change in cytochrome a(3), and after this, (ii) a change in the molecular environment of the cytochrome a. The latter effect, possibly a decrease in the hydrophobicity of the haem pocket, requires that the ligands on cytochrome a(3) have a bulky and partially hydrophobic character.     

Action Spectra and the Role of Carotenoids in Photosynthesis

The action spectrum of photosynthetic (Vprochtetion was determined with ehloro-plast suspensions and whole leaves. Owing to a dominating influence of light scattering the action spectrum shows a much more uniform absorption of the incident light than would be expected from the absorption spectrum in direct light (0° deviation). The comparatively strong photosynthesis in green is probably further aided by the light reaction between cytochrome f and chlorophyll in which also the cyto-chromes act as pigments. The whole region 400 to about 900 mn is photosynthetically active with an obvious tendency to linear relation between the intensity of incident radiation ami photosynthetic Co-production, certain deviations hereby caused by specific absorption or activation. The results illustrate the participation of carotenoids in the primary energy conversion of photons to activated electrons and the role of at least two light reactions in the photosynthetic cycle. The reducing power of illuminated (3-carotene was demonstrated in vitro in its effect on ferredoxin. The steady state situation of pigments and enzymes is discussed.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

One-electron reactions in biochemical systems as studied by pulse radiolysis. VI. Stages in the reduction of ferricytochrome c

When ferricytochrome c at pH about 9 is reduced by hydrated electrons and/or CO₂^?, it gives rise to an unstable form of ferrocytochrome c whose absorption spectrum, particularly in the Soret region, differs from that of normal ferrocytochrome c. This form changes intramolecularly (life-time about 0.1 s at ambient temperature) to yield normal ferrocytochrome c, and by 0.5 s the change in absorption spectrum in the range 225–600 nm produced by e^?_aq and/or CO₂^? is identical to the final change produced by reduction with an equivalent amount of sodium dithionite. This shows that both e^?_aq and CO^?₂ reduce cytochrome c with practically 100% efficiency. In the range 600–800 nm the spectrum of the unstable form is the same as that of normal ferrocytochrome c, both having small absorptions at 695 nm as compared with ferricytochrome c. As the unstable form disappears however a further loss of absorption at 695 nm occurs. This is taken to imply that the unstable form decays to a second unstable form which then rapidly donates an electron to the unchanged neutral form of ferricytochrome c, so reducing absorption in the 695 nm band. Subsequent to this process the absorption in the 695 nm band increases over a period of minutes owing to re-equilibration between the neutral and alkaline formes of ferricytochrome c. Between pH 7 and 10 the effect of pH on the absorption changes is consistent with the hypothesis of a second unstable form of ferrocytochrome c. Additional phenomena arise in more alkaline solutions. The rates of the various unimolecular processes are thought to be determined by the rates of change of conformation of the protein parts of the molecule following the change in oxidation state.     

Action Spectrum of Coccolith Formation

The action spectrum of coccolith formation in Coccolithus huxleyi was determined by measuring the uptake of carbon-14 in coccoliths in four-hour experiments as a function of light intensity at each of seven wavelengths. An action spectrum | of photosynthetic carbon assimilation was obtained at the same time. The coccolith action spectrum had peaks at wavelengths of about 440 nm and 670 nm. probably corresponding to the regions of maximum cellular absorption and carbon assimilation. However, blue light appeared to be relatively more efficient in coccolith formation than in carbon assimilation. The results suggest that light-dependent coccolith formation may be catalyzed by two photochemical reactions, one mediated by chloroplast pigments and the other by some pigment absorbing specifically in the blue part of the spectrum.     

Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. III. Light-Induced Absorbance Changes in Chloroplast Fragments of the Wild Type and Mutant Strains

Light-induced absorbance changes were investigated in chloroplast fragments of wild type Chlamydomonas reinhardi and 5 different mutant strains having impaired photosynthesis. Two absorbance changes were detected, 1 having a maximum at 553 nm and the other at 559 nm. The component exhibiting the 553 nm change is a cytochrome similar to cytochrome f from higher plant chloroplasts. The component exhibiting the 559 nm change has the properties of a cytochrome similar to cytochrome b(3). Two of the mutant strains (ac-115 and ac-141) were found to lack the 559 cytochrome and light induced only the oxidation of the 553 cytochrome. A third mutant strain (ac-206), previously shown to lack the 553 cytochrome, exhibited only the light-induced reduction of the 559 cytochrome. A fourth mutant strain (ac-208), shown to lack plastocyanin, exhibited absorbance changes attributable to both cytochromes. However, light was capable of inducing the reduction of the 559 cytochrome but not its oxidation. On the other hand, light induced the oxidation of the 553 cytochrome but not its reduction.These observations are discussed in terms of the series formulation for photosynthetic electron transport in which the 559 cytochrome is reduced by system II and transfers electrons via the component affected in ac-21 to the 553 cytochrome. Accordingly, system I sensitizes the oxidation of the 3 components of the electron transport chain.     

Effect of dibromothymoquinone on photosynthetic electron transport]

The effect of dibromothymoquinone on photosynthetic electron transport in pea dependent on concentration was studied. Dibromothymoquinone inhibited general electron transport from water to NADP+ in isolated chloroplasts and ethiochloroplasts and the electron transfer via plastoquinone and cytochrome f in the leaves and isolated plastids. At all concentrations studied dibromothymoquinone significantly affected the absorption changes at 590 nm in the ethiochloroplasts associated with plastocyanine photoreactions. Possible location of electron carriers in the photosynthetic electron transport chain is discussed.     

Cytochrome oxidation in bacterial photosynthesis

In this paper we propose that the reduction of the bacteriochlorophyl dimer cation (P⁺) by cytochrome c in the photosynthetic bacteria Rps. viridis and Chromatium vinosum proceeds via two parallel electron transfer (ET) processes from two distinct cytochrome c molecules. The dominating ET process at high temperatures involves the activated oxidation of the high-potential cytochrome c at closest proximity to P, while the dominating low-temperature process involves activationless ET from a low-potential cytochrome c, which is further away from P. The available data for the effects of blocking the low-potential cytochrome c on ET dynamics are consistent with this model, which results in reasonable nuclear reorganization and electronic coupling parameters for the parallel cytochrome oxidation processes. The lack of universality in the cytochrome oxidation in reaction centres of various bacteria is emphasized.     

Cytochromebc purified from the methanogenMethanosarcina barkeri

Cytochromebc was partially purified from the methanogen,Methanosarcina barkeri. The cytochrome was composed of three subunits with molecular masses of 23.4, 20.9, and 9.1 kDa, respectively, and the 23.4 kDa subunit contained haemc. The absorption spectrum of cytochromebc showed a peak at 411 nm in the oxidized form, and peaks at 554, 524, and 422 nm in the reduced form. The cytochrome reacted with CO, and its low temperature absorption spectrum showed the peak at 552 nm with a shoulder at 557 nm.     

The effect of detergent on the structure and composition of chlorosomes isolated from Chloroflexus aurantiacus

Isolated chlorosomes, treated with the detergent lithium dodecyl sulfate (LDS), can be separated into two green fractions by agarose gel electrophoresis. One fraction contains chlorosomes with a full complement of proteins and antenna BChl c absorbing at 740 nm, but with a more spherical form than the normal ellipsoid shape observed in control chlorosomes. The second fraction was completely devoid of proteins but had a similar absorption spectrum. Electron micrographs of the protein-free fraction indicated the presence of stain-excluding spheres with overall dimensions resembling those of intact chlorosomes (40–100 nm). These spheres are probably micelles of BChl c liberated from the chlorosomes during the detergent treatment, since similar structures could be produced when purified BChl c, dissolved in 1-hexanol, was dispersed in buffer, producing an aggregate absorbing at 742 nm. These results suggest that the chlorosome proteins are not required to produce an arrangement of BChl c chromophores which gives rise to a 740 nm absorption peak resembling that of intact chlorosomes. It seems probable, however, that proteins have a role in determining the overall shape of the chlorosome. Treatment with cross-linking reagents did not prevent the detergent-induced changes in chlorosome morphology.Abbreviations BChl bacteriochlorophyll - DSP dithiobis-succinimidyl-2-propionate - EM electron microscopy - LDS lithium dodecyl sulfate - MGDG monogalactosyl diacylglycerol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis