Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi VI. Electron Transport in Mutant Strains Lacking Either Cytochrome 553 or Plastocyanin

A mutant strain of Chlamydomonas reinhardi, ac-206, lacks cytochrome 553, at least in an active and detectable form. Chloroplast fragments of this mutant strain are inactive in the photoreduction of NADP when the source of electrons is water, but they are active when the electron source is 2,6-dichlorophenolindophenol and ascorbate. The addition of either cytochrome 553 or plastocyanin, obtained from the wild-type strain, has no effect upon the photosynthetic activities of the mutant strain. Cells of the mutant strain lack both the soluble and insoluble forms of cytochrome 553, but they possess the mitochondrial type cytochrome c. Thus, the loss of cytochrome 553 appears to be specific.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. V. Purification and Properties of Cytochrome 553 and Ferredoxin

Cytochrome 553 and ferredoxin were isolated and purified from acetone powders prepared from intact cells of the wild-type strain of Chlamydomonas reinhardi. Purification was achieved by ion exchange chromatography on DEAE cellulose and gel filtration on Sephadex G-75.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. VII. Photosynthetic Phosphorylation by a Mutant Strain of Chlamydomonas reinhardi Deficient in Active P700

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor.

These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

     

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.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi: VIII. The 520 nm Light-induced Absorbance Change in the Wild-type and Mutant Strains 1

The 520 nm light-induced absorbance change in wild-type and 4 mutant strains of Chlamydomonas reinhardi was investigated. In the wild-type strain the absorbance change is composed of at least 2 components, P520 I and P520 II, sensitized by Systems I and II respectively. Some of the properties of these components can be studied by using the appropriate photosynthetic mutant strain. A group of mutant strains modified in the photochemical complex of System II shows only the P520 I absorbance change, whereas a mutant strain deficient in active P700 exhibits only the P520 II absorbance change. The possible relationship between these absorbance changes and the photosynthetic electron transport pathway is discussed.     

Transport of urea at low concentrations in Chlamydomonas reinhardi.

Urea transport into the unicellular green alga Chlamydomonas reinhardi was investigated to further our understanding of controls operating on urea catabolism in this organism. Transport into cells grown with acetate and deprived of ammonia is a saturable process, mediated by at least two systems operating maximally at different external urea concentrations. The lower concentration system, with an apparent Km for urea of 5.1 micron, was the object of detailed study. Transport of urea from a saturating concentration (57 micron) into ammonia- and acetate-grown cells freshly suspended in ammonia-limited medium was not detected. Upon further culturing in the absence of ammonia, derepression occurred with transport ability, first appearing at about 1 h , reaching a maximum at about 2 h, and maintaining this maximum at least 5 h. In contrast to this, CO2-grown cells became derepressed more slowly, and maximum transport ability was not maintained. Addition of ammonia or methylamine (5 mM) during nitrogen deprivation prevented further increases in transport ability and caused loss of previously acquired transport ability. Cycloheximide (10 microng/ml) had a similar effect. Energy uncouplers or dark, anaerobic conditions depressed transport. By these criteria, transport from low urea concentrations is mediated by a process that requires protein synthesis and activation by cellular energy, and the process has a rapid rate of turnover and of deactivation by ammonia.     

Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi.

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.     

Energy and Electron Transfer Systems of Chlamydomonas reinhardi: II. Two Cyclic Pathways of Photosynthetic Electron Transfer in the Pale Green Mutant

Light- and oxygen-induced changes of cytochromes f, b₅₆₃, and b₅₅₉ and ferredoxin-flavoprotein were studied by a double beam spectrophotometer with combinations of inhibitors and lowered temperatures in the whole cells of the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). At room temperature, the steady state changes of cytochrome f and ferredoxin-flavoprotein are small, but at low temperature slightly above 0 C, they are clearly defined. Phenylmercuric acetate inhibits photoreduction of ferredoxin-flavoprotein and cytochrome f simultaneously but not that of cytochrome b₅₆₃. 2-Heptyl-4-hydroxyquinoline-N-oxide shows a crossover point between cytochromes f and b₅₆₃ and partially inhibits photoreduction of cytochrome f. Two cyclic pathways operating in C. remhardi are postulated: (a) photosystem I → x → b₅₆₃ → f → photosystem I; and (b) photosystem I → x → ferredoxin-flavoprotein → f → photosystem I.     

Energy and Electron Transfer Systems of Chlamydomonas reinhardi. I. Photosynthetic and Respiratory Cytochrome Systems of the Pale Green Mutant

The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b₅₆₃ and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b₅₅₉ is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

Morphological and Photosynthetic Properties of Digitonin-treated Chloroplast Membranes from the Wild-type and ac-5 Strains of Chlamydomonas reinhardi

Chloroplast membranes of wild-type Chlamydomonas reinhardi, treated with digitonin, yield photosystem II-rich and photosystem I-rich fractions; this fractionation is accompanied by a separation of stacked (grana) lamella from unstacked (stroma) lamellae. Poor fractionation of the photosystems occurs when the treated chloroplast membranes derive from the ac-5 strain grown mixotrophically, whereas good fractionation occurs with ac-5 cells grown phototrophically; the mixotrophic cells possess only unstacked membranes, whereas the phototrophic cells possess stacked membranes. We concluded that digitonin fractionation is dependent on the stacked membrane configuration.     

Photosynthetic Properties of ac-31, a Mutant Strain of Chlamydomonas reinhardi Devoid of Chloroplast Membrane Stacking

A pale-green mutant strain of Chlamydomonas reinhardi, ac-31, is characterized by the absence of any stacking of its chloroplast membranes. The capacity for photosynthetic electron transport, phosphorylation, and CO₂ fixation in ac-31 is substantial, and it is concluded that these photosynthetic activities occur within the single membrane. The photosynthetic capacities of wild type and ac-31 as a function of increasing light intensity are compared. Saturation is attained at higher light intensities in ac-31, and the kinetics of the 2 sets of curves are distinctly different. The possibility that energy transfer is enhanced by membrane stacking is suggested by these results. The repeatedly-observed correlation between reduced stacking and disfunctional Photosystem II activities is discussed in view of the observation that ac-31 has no stacking but retains a functional Photosystem II.     

The Effect of Manganese on Chloroplast Structure and Photosynthetic Ability of Chlamydomonas reinhardi

Manganese deficiency was induced in mixotrophically-grown cultures of the green alga Chlamydomonas reinhardi and the effects of this deficiency on photosynthetic activity and cell structure were investigated. Manganese-deficient cells are unable to carry out, at normal rates, photosynthetic reactions involving system II of the photosynthetic electron transport chain. Reactions requiring only system I are not inhibited. Normal system II activity returns within 2 hr in the light following addition of manganous ions to deficient cells.Significant structural alterations for deficient cells were observed in the chloroplast, in the arrangement of discs into stacks. Stacking distributions for normal, deficient, and recovered cells were quantitatively characterized and compared by the introduction of an experimentally-defined distribution function and its attendant parameters. The results provide evidence for a role for manganese in maintaining chloroplast structure.     

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

In the photosynthetic light reactions of plants and cyanobacteria, plastocyanin (Pc) plays a crucial role as an electron carrier and shuttle protein between two membrane protein complexes: cytochrome b₆f (cyt b₆f) and photosystem I (PSI). The rapid turnover of Pc between cyt b₆f and PSI enables the efficient use of light energy. In the Pc-cyt b₆f and Pc-PSI electron transfer complexes, the electron transfer reactions are accomplished within <10⁻⁴ s. However, the mechanisms enabling the rapid association and dissociation of Pc are still unclear because of the lack of an appropriate method to study huge complexes with short lifetimes. Here, using the transferred cross-saturation method, we investigated the residues of spinach (Spinacia oleracea) Pc in close proximity to spinach PSI and cyt b₆f, in both the thylakoid vesicle–embedded and solubilized states. We demonstrated that the hydrophobic patch residues of Pc are in close proximity to PSI and cyt b₆f, whereas the acidic patch residues of Pc do not form stable salt bridges with either PSI or cyt b₆f, in the electron transfer complexes. The transient characteristics of the interactions on the acidic patch facilitate the rapid association and dissociation of Pc.     

Models of Photosynthetic Electron Transport

Biophysics - Energy transduction reactions in the photosynthetic membrane are a primary step in storing solar energy to be used later in biosynthetic and other processes in living systems. This...     

Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C₄ dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.