Richard Malkin

Chlorophyll a/b proteins of Photosystem I

The chlorophyll a/b‐protein complex (LHCPI) associated with Photosystem I (PS I) has been isolated from spinach thylakoids and further fractionated into two chlorophyll‐containing complexes by sucrose gradient centrifugation. The lighter fraction contains two polypeptides with relative molecular masses of 23 and 22 kDa and has been designated as LHCPIa. The denser fraction is enriched in a 20 kDa polypeptide and has been named LHCPIb. Both fractions have a chlorophyll a/b ratio of 3.5 ± 0.5. The absorption spectra and 77 K fluorescence emission spectra of the fractions show distinct characteristics with LHCPIb having a fluorescence maximum at 730 nm at 77 K while LHCPIa shows a maximum at 680 nm. The optical activities of the chlorophyll a/b complexes and the antenna‐depleted PS I have been examined by circular dichroism (CD) in the near‐UV and visible regions of the spectrum. All the LHCPI complexes show strong CD signals at 648, 485 and 340 nm which are absent in the antenna‐depleted PS I complex.

The reconstitution of clostridial ferredoxin.

Previous experiments from this laboratory (Lovenberg, et al., 1963; Malkin & Rabinowitz, 1966) indicated that when bacterial ferredoxin is treated with a mercurial, the color of the protein is bleached, and both the iron and the acidlabile sulfide of the protein are released. Regeneration of ferredoxin could be accomplished by the addition of 2-mercaptoethanol to the bleached reaction mixture. Apoproteins were isolated by chromatography of the reaction mixture on DEAE-cellulose or passage over Sephadex. However, it was not possible to reconstitute ferredoxin from the isolated proteins by the addition of iron and inorganic sulfide under any conditions tested. In the present communication, we wish to describe two methods for the preparation and isolation of apoferredoxins, and their use in the reconstitution of ferredoxin by the addition of ferrous ions and 2-mercaptoethanol and ferrous ions, sodium sulfide, and 2-mercapto-ethanol respectively. The reconstituted materials are identical to native ferredoxin with respect to enzymic activity, acid-labile sulfide and iron content, and ultraviolet and visible absorption spectra.

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

Abstract An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg2+. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS IIα. (5) An Em = − 10 mV has been measured at pH 7.8 for the primary electron acceptor Qα of PS IIα. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll a b- protein . In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.

Identification of a g = 1.90 high-potential iron-sulfur protein in chloroplasts

Abstract A new bound iron-sulfur protein has been identified in spinach chloroplasts. In the reduced form, this protein has an electron paramagnetic resonance spectrum at 20°K with g -values of 2.02 and 1.90. The midpoint oxidation-reduction potential (E m ) of the protein, which is pH-independent, is +290 mV. These properties are similar to those of the “Rieske” g = 1.90 iron-sulfur protein of mitochondrial Complex III.

The structure of spinach Photosystem I studied by electron microscopy

The structure of three types of Photosystem I (PS I) complex isolated from spinach chloroplasts was studied by electron microscopy and computer image analysis. Molecular projections (top views and side views) of a native PS I complex (PSI-200), an antenna-depleted PS I complex (PSI-100) and the PS I reaction center complex (CPI) were analyzed. The overall structure of the native PS I complex was found to be a disk with dimensions (corrected for attached detergent) of 16 × 12 nm in the plane of the membrane and a height of 6.8 nm. The PSI-100 and CPI complexes gave much smaller projections but these were similar in shape and size to those of the previously analyzed cyanobacterial PS I complex. The arrangement of the subunits within the native PS I complex is discussed. It is concluded that a shell of about eight light-harvesting complex (LHCI) subunits attached to the PSI-100 complex fits the dimensions and shape of the PSI-200 complex.

Quantitative EPR studies of the primary reaction of Photosystem I in chloroplasts

Abstract Quantitative electron paramagnetic resonance studies of the primary event associated with Photosystem I in chloroplasts have been carried out at 25 °K. After illumination of either whole chloroplasts or Photosystem I subchloroplast fragments (D-144) with 715-nm actinic light at 25 °K, equal spin concentrations of oxidized P700 and reduced bound iron-sulfur protein (bound ferredoxin) have been measured. Quantitative determination of the concentration of these two carriers by EPR spectroscopy after illumination at low temperature indicates that Photosystem I fragments are enriched in P700 and the bound iron-sulfur protein as compared with unfractionated chloroplasts. These results indicate that P700 and the bound iron-sulfur protein function as the donor-acceptor complex of chloroplast Photosystem I.

Isolation of the rieske iron-sulfur protein from the cytochrome b6/f complex of spinach chloroplasts

The organization of cytochrome b/c complexes in electron-transfer chains of mitochondria [1 ], chloroplasts [2,3] and photosynthesizing bacteria [4], is strikingly similar. In addition to cytochrome b and c~ (called cytochrome f in chloroplasts), an essential component of these complexes is a Fe-S center with a midpoint potential of ~300 mV [ 1,3-7]. It was first isolated from beef heart mitochondria in [5], and has been called the Rieske Fe-S protein thereafter. In [8,9] this Fe-S protein has been purified in active form, reconstituting electron transport from succinate to cytochrome c in depleted preparations of the succinate-cytochrome c reductase complex from beef heart mitochondria. The Rieske Fe -S center has been detected in chloroplasts [6] and found in a highly purified, enzymatically active preparation of cytochrome b6/fcomplex [3]. Here, we report on the identification of the second smallest polypeptide of the complex as the Rieske Fe -S protein. It can be isolated from the complex retaining the characteristic EPR signals. The deficient complex is inactive in plastoquinol-plastocyanin-oxidoreductase activity and lacks the EPR signals of the Fe-S center. An antibody, prepared against the isolated Rieske Fe-S protein inhibits the oxidoreductase activity of the intact complex. The effects of the inhibitory plastoqkfinone analogue, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, (DBMIB) which has been found to shift the g-value of the EPR signals of the Rieske Fe -S center in chloroplast membrane [10,11 ], on the EPR-spec-

Characterization of an isolated chloroplast membrane Fe-S protein and its identification as the photosystem I Fe-SA/Fe-SB binding protein

An iron‐sulfur protein of approx. 9 kDa has been isolated from spinach chloroplast membranes. Based on iron and sulfide content (∼900 nmol Fe and S2− per mg protein), optical absorbance and low‐temperature EPR spectra, it appears that this protein contains 8Fe and 8S2− in two [4Fe◀4S] clusters. The protein cross‐reacts with an antibody raised against a 9 kDa PS I subunit previously identified as the psaC (frxA) gene product. This identity was confirmed by the N‐terminal amino acid sequence of the isolated Fe◀S protein. It is concluded that the isolated Fe◀S protein binds PS I centers Fe◀SA and Fe◀SB and that each of these centers contains 4Fe and 4S2−.

On the function of two vitamin K1 molecules in the PS I electron acceptor complex

Various PS I preparations from higher plants and cyanobacteria are found to contain vitamin K1, or phylloquinone, in a stoichiometry of 2 vitamin K1/P700. Extractions of lyophilized PS I preparations with organic solvents led to removal of one molecule of vitamin K1 but conditions have not been established for the removal of the second quinone. The quinone‐depleted PS I complex ( ~ 1 vitamin K1/P700) was fully active in transferring electrons at both physiological and cryogenic temperatures, indicating one vitamin K1 molecule was not required for these activities.

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X‐100. CP1a has a Chl a/b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X‐100 PS I complex (Chl a/b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a/b‐proteins of the main light‐harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X‐100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a/b‐proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.