Stephen Lien

Isolation and characterization of a subchloroplast particle enriched in iron-sulfur protein and P700

Abstract Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by differential centrifugation and Sephadex G-200 and DEAE Bio-Gel A chromatography results in isolation of two distinct particles containing iron-sulfur protein. The first is a P700-containing particle that contains 8–10 g-atoms each of nonheme iron and labile sulfide and 23 chlorophylls per mole of P700 but no detectable levels of chlorophyll b or cytochromes f , b 6 , or b 559 . The second particle exhibits no P700 activity but does contain cytochromes f and b 6 in equimolar amounts in addition to 2–4 g-atoms each of nonheme iron and labile sulfide per mole of cytochrome f . Virtually all the nonheme iron and labile sulfide present in spinach thylakoids is accounted for in these two particles. Further treatment of the P700-enriched particle with urea and potassium ferricyanide causes a time-dependent loss of labile sulfide in concert with the loss of photoactive P700. In contrast, the environmental integrity of P700 is unaffected by this treatment since there is no corresponding absorbance change in the chemical oxidized-minus-reduced difference spectrum. Control levels of labile sulfide are reestablished in the depleted particles by overnight treatment with dithiothreitol. Pretreatment of the depleted particles with cyanide prevented the recovery of labile sulfide by preincubation with dithiothreitol. In accord with these data, a mechanism is invoked for the oxidation of labile sulfide to zero-valence sulfur, S 0 , in the bound iron-sulfur proteins, which results in destruction of the iron-sulfur core.

Regulation of chlorophyll synthesis in photosynthetic bacteria

Energy-transducing membranes of the nonsulfur purple photosynthetic bacteria are known to contain several species of bacteriochlorophyll (BChl) complexes. The reaction-centre complex (rc-BChl) is the locus of the charge separation that provides the “poles” of the photochemical electron transport system, whereas the other complexes serve lightharvesting functions. This report summarizes an investigation of the general features of the control mechanisms governing synthesis of the several chlorophyll complexes inRhodopseudomonas capsulata. The results obtained indicate a close biosynthetic association between rc-BChl and one of the light-harvesting chlorophylls (complex I). Regulation of synthesis of light-harvesting complex II (during anaerobic photosynthetic growth) appears to be relatively independent, and intimately related to the “energy state” of the cell. Chlorophyll synthesis inR. capsulata cells growing aerobically in darkness was also studied. The presence of functional photosynthetic units in dark-grown cells, of very low BChl content, was clearly evidenced by demonstration of: the potentiality for resumption of anaerobic photosynthetic growth, light-induced oxidation of cytochrome552in vivo, and high photophosphorylation capacity (relative to BChl) of membrane fragments from such cells. Synthesis of light-harvesting BChl complex II is particularly inhibited in cells growing in darkness with respiratory phosphorylation as the source of energy, and it is suggested that this complex is a primary “target” of the biosynthetic control devices activated by change of light intensity or presence of molecular oxygen during growth of nonsulfur purple bacteria.

Interaction of plastocyanin and P700 in PSI reaction center particles from C. reinhardi and spinach

Abstract Highly resolved Photosystem I (PSI) reaction center particles, solubilized from spinach and C. reinhardi chloroplasts with Triton X-100, catalyze very poorly the reduction of P700 by ascorbate at mildly acidic to alkaline pHs (pH 5–8). (Photooxidation of P700 means conversion of P700 to P700+; dark reduction of P700 is the reverse reaction.) The interaction between plastocyanin (PC) and P700 is assessed by measuring the stimulation of the rate of P700 reduction upon addition of PC at submicromolar concentrations. At a ratio of 2.6 PC per P700, about 20- to 25-fold stimulation of the rate of P700 reduction was observed in the presence of MgCl2 or CaCl2. Efficient interaction between PC and P700 occurred at near neutral pH (optimal pH = 6.6–6.8) and exhibited a preferential requirement for divalent metal cations. Monovalent cations are less effective in promoting transfer of electrons from PC to P700. At alkaline pHs (greater than 8.5) an accelerated rate of the PC-independent reduction of P700 was observed. Further, the PC-independent reaction at pH 9.4 showed a strong requirement for divalent cations. In contrast, monovalent cations are only partially effective in promoting the PC-independent reduction of P700 by ascorbate.

On the role of membrane-bound ADP and ATP in photophosphorylation in chloroplast membranes

The role of CFr firmly-bound nucleotides in photophosphorylation is of current and widespread interest and importance; especially as regards the proposal that conformational changes of the membrane-bound protein result in altered binding affinities for the substrates, ADP, Pi and ATP [ 1,2]. In addition, a lightdependent and uncoupler-sensitive exchange of nucleotides into membrane-bound CFr was recently discovered by Harris and Slater [3]. The relationship between this type of exchange reaction and ATP formation in chloroplasts was further investigated by Strotmann et al. [4] and Magnusson and McCarty [S] . Energization of thylakoid membranes in the presence of labeled ADP or ATP resulted in the incorporation of ADP into CFr . The nucleotide bound to CF, in this manner appears for the most part to be associated with one of the two larger subunits of CFr . Recently, we reported on the light-dependent phosphorylation of CFr-bound ADP and attempted to correlate this activity with the phosphorylation of free ADP [6] . Using short term illumination (2 3 s), about one mole of bound ADP per mole of CFr was phosphorylated to yield an equivalent amount of [

Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C reinhardtii.

“P] ATP. These studies have been further extended using short-term (2 30 ms), saturating illumination to attempt to compare the kinetics characteristic of phosphorylation of bound and free ADP. In this communication, it is shown that the rate of phosphorylation of bound nucleotide: (1) Is much lower than that observed with free ADP.

On the reactivity of oxygen with photosystem I electron acceptors

Abstract An anaerobic incubation period of varying duration is required to induce hydrogenase activity in C. , reinhardtii . Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.

Determination of acid-labile sulfide and zero-valence sulfur in subchloroplast particles in the presence of sodium dodecyl sulfate

The reduction of molecular oxygen as a consequence of photosynthetic electron transport was initially demonstrated [ 1,2] where oxygen was shown to serve as a Hill oxidant. Subsequently, it was recognized that the major site for reduction of oxygen is via photosystem I 13-51. In the absence of an artificial electron donor system, oxygen reduction by illuminated broken chloroplasts (poisoned with KCN to inhibit catalase) was shown [S] to be saturated by an oxygen tension equivalent to 4% (partial pressure) oxygen in nitrogen. Further, an app. Km of 5 FM was measured [S] for oxygen with the broken chloroplast preparation. Since soluble ferredoxin was assumed to be absent, the app. Km was suggested [S] to reflect the affinity of the PSI reductant (other than ferredoxin) for molecular oxygen. There is now substantial evidence to indicate that the endogenous Mehler reaction (oxygen reduction to produce hydrogen peroxide) involves a univalent reduction of oxygen and formation of a superoxide free radical [6,7]. The photoreduction of cytochrome c mediated by both ferredoxin and by superoxide radical was studied [7]. Photoreduction of cytoc~ome c, under aerobic conditions and in the presence of 0.5-I I.IM

Electron Transport in Chloroplasts

Abstract A method for the determination of acid-labile sulfide and zero-valence sulfur (by conversion to sulfide with dithiothreitol) in subchloroplast particles in the presence of sodium dodecyl sulfate is described. A procedure to eliminate interfering sulfide production from dithiothreitol alone is provided. After color development, methylene blue is separated from interfering colored substances by cellulose phosphate column chromatography. The method requires no special equipment and allows determination of acid-labile sulfide in the micromolar range.

On the identity of photo- and oxidative phosphorylation coupling factors in Rhodopseudomonas capsulata

The result of photosynthetic electron transport in green plants is the evolution of molecular oxygen and the formation of the assimilatory power necessary for the conversion of carbon dioxide into cellular material at the expense of light energy. The currently accepted representation of photosynthetic electron transport as the cooperative interaction of two light reactions originated with Hill and Bendall (1960). A modified schematic representation of their hypothesis is presented in Figure 1. Their formulation was proposed primarily to account for three major experimental observations: First, the decline in efficiency of photosynthesis at long wavelengths (λ>685 nm) and the synergistic effect of shorter wavelengths on far red illumination (Emerson and Lewis, 1943; Blinks, (1957). Secondly, the presence in green tissues of two cytochromes, cytochrome f and b 6 (564) whose characteristic potentials (E m, 7) differed by about 0.4 V and their light-induced absorbance changes (Hill, 1965). Lastly, the stimulation of electron flow to NADP+ when ATP formation occurred concurrently (Arnon et al., 1958; Davenport, 1959).

A uniparentally inherited mutation affecting photophosphorylation in Chlamydomonasreinhardi

Abstract The nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata can obtain energy for growth either by anaerobic photophosphorylation or dark oxidative (aerobic) phosphorylation. Earlier evidence for the in vitro interchangeability of phosphorylation coupling factor preparations from dark (aerobically) grown and photosynthetically grown cells in both kinds of energy conversion has been extended, through closer study of the factor obtained from aerobic cells cultivated under conditions which permit only very slight development of the photosynthetic apparatus. It is concluded that the same protein factor couples phosphorylation to both respiratory and photosynthetic electron transport.