Anthony San Pietro

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.

Ferredoxin reducing substance (FRS) from spinach

Abstract The requirement for a heretofore unidentified chloroplast component for NADP photoreduction by chloroplast fragments is established. This component, termed “ferredoxin reducing substance (FRS),” is required also for the photoreduction of methyl viologen and, in the reduced form, serves as the reductant for the dark reduction of NADP or ferredoxin. These data suggest that FRS is the primary acceptor for photosystem 1 of photosynthesis.

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.

Interchangeability of phosphorylation coupling factors in photosynthetic and respiratory energy conversion.

The nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata can obtain energy for growth either by anaerobic photophosphorylation or dark oxidative (aerobic) phosphorylation. Successful resolution of phosphorylation coupling factors from energy-converting membranes of this bacterium permitted tests for reciprocal function of such protein factors in oxidative-and photophosphorylation processes. Evidence was obtained for the interchangeability of coupling factor preparations from dark-grown and photosynthetically grown cells in both kinds of energy conversion.

Studies on the photoreduction of ferredoxin and the ferredoxin-NADP reductase flavoprotein by chloroplasts fragments: Effect of pyrophosphate

Abstract The extinction coefficients of the different oxidation states of the chloroplast flavoprotein, ferredoxin-NADP reductase, have been determined in the spectral region from 600 to 400 nm. The photoreduction of the flavoprotein by chloroplast fragments produces the semiquinone, fp · FADH•, and not the fully reduced form of the enzyme, fp · FADH2. The photoreduction of the flavoprotein, and of NADP, is inhibited by inorganic pyrophosphate. This metabolite does not affect the photoreduction of ferredoxin, and therefore it is a specific inhibitor at the level of the oxidation of reduced ferredoxin in the electron transport system of chloroplasts. These observations are discussed in relation to the function of ferredoxin, flavoprotein, and pyrophosphate in photosynthesis.

Preparation and characterization of a chemically modified plastocyanin

Abstract The reaction of plastocyanin with tetranitromethane results in the nitration of only one of the three tyrosyl residues present in the protein. The modification does not affect the blue copper chromophore as both the characteristic visible spectrum of the chromophore and the redox potential of the protein are unchanged. Photochemical assays show that the modified plastocyanin is fully active in the reduction of photooxidized P700 and in the photooxidation of cytochrome f . The p K of the nitro-tyrosyl residue is about 7.3 indicating that the modified residue may be located in a negatively charged environment. Examination of the recently published X-ray structure of poplar plastocyanin suggests that Tyr-80 would be a likely candidate for the site of modification.

Determination of acid-labile sulfide in subchloroplast particles containing Triton X-100

Abstract A method has been devised whereby measurement of the acid-labile sulfide content of spinach subchloroplast particles is free of interference resulting from the presence of Triton X-100. Quantitative extraction into an organic solvent allows accurate detection of micromolar concentrations of sulfide.

Chemical modification of spinach ferredoxin: Evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction

Abstract Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome c . The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.

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 [

Ferredoxin-reducing substance (FRS) from spinach: II. Separation and assay

“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.