M. Rögner

Refined purification and further characterization of oxygen-evolving and Tris-treated Photosystem II particles from the thermophilic Cyanobacterium synechococcus sp.

Highly active, monomeric and dimeric Photosystem II complexes were purified from the thermophilic cyanobacterium Synechococcus sp. by two sucrose density gradients, and the size, shape and mass of these complexes have been estimated (Rogner, M., Dekker, J.P., Boekema, E.J. and Witt, H.T. (1987) FEBS Lett. 219, 207–311). (1) Further purification could be obtained by ion-exchange chromatography, by which the 300 kDa monomer could be separated into a highly active, O2-evolving fraction, and a fraction without O2-evolving capacity, which has lost its extrinsic 34 kDa protein. Both showed very high reaction center activities as measured by the photoreduction of the primary quinone acceptor, QA, at 320 nm, being up to one reaction center per 31 Chl a molecules. (2) Tris-treatment yielded homogeneous 300 kDa particles which had lost their extrinsic 34 kDa polypeptide. Electron microscopy of this complex revealed very similar dimensions compared to the oxygen-evolving 300 kDa particle, except that the smallest dimension was decreased from about 6.5 nm to about 5.8 nm. This difference is attributed to the missing extrinsic 33 kDa protein, and the smallest dimension is attributed to the distance across the membrane. (3) Experiments are presented, allowing an estimation for the contribution of detergent to the other dimensions being about 2 × 1.5 nm for dodecyl β-d-maltoside. This leads to dimensions, corrected for detergent size, of 12.3 × 7.5 nm for the monomeric form of PS II and 12 × 15.5 nm for the dimeric form. (4) From some extracts a 35 kDa, chlorophyll-binding complex could be isolated which lacks the characteristic absorbance changes of QA and of Chl aII (P-680) and is therefore supposed to be a light-harvesting complex of cyanobacteria. (5) A model for the in vivo organization of PS II in cyanobacteria is discussed.

Mono-, di- and trimeric PS I reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus sp.: Size, shape and activity

Abstract Photosystem I preparations from the cyanobacterium Synechococcus sp. were treated with high concentrations of Tris and octyl glucoside at alkaline pH and elevated temperature. A sucrose density gradient yielded three pigment-protein complexes; these were further purified on a HPLC anion-exchange column. In contrast to SDS-PAGE under non-denaturing conditions, this method produces homogeneous, highly active PS I particles in large quantities using mild solubilization conditions. Gel-filtration HPLC, SDS-PAGE, antenna Chl Chl a 1 ratio and electron microscopy images suggest that the three complexes represent monomeric, dimeric and trimeric forms of a minimal reaction center I unit. The size of this monomeric complex is 15.3 × 10.6 × 6.4 nm (length × width × height) as determined by electron microscopy. The apparent molecular mass - including 65 antenna chlorophylls per Chl a1, but excluding the detergent shell - is estimated by three different methods to be (235 ± 25) kDa.

Refined analysis of the trimeric structure of the isolated Photosystem I complex from the thermophilic cyanobacterium Synechococcus sp.

The trimeric structure previously evaluated from isolated Photosystem I (PS I) reaction-center complex of the thermophilic cyanobacterium Synechococcus sp. was studied in more detail by electron microscopy and computer image analysis. Molecular projections (1970 top views and 457 side views) were selected from electron micrographs and subsequently aligned to different references. The top views were submitted to a multivariate statistical classification procedure. Two types of top view, whichdiffer in handedness and represent face-up and face-down oriented molecules, could be separated. The classification shows that the PS I trimeric complex consists of three very similar, if not identical, units. The trimer has the shape of a disk with a diameter of 19 nm and a thickness of 6.5 nm. The monomers within the trimer have an asymmetric shape. Possible arrangements of the two major subunits within the monomer are discussed.

Field-driven ATP synthesis by the chloroplast coupling factor complex reconstituted into liposomes

Since the development of the chemiosmotic theory [l] there has been a continuous effort to experimentally simplify the energy-transducing apparatus in chloroplasts as well as in mitochondria and bacteria. This has been done by structural or functional isolation and characterization of partial reaction sequences. One direction of functional isolation involved replacing the electron transportinduced energization of the thylakoid membrane by an energization with an artificially generated proton gradient, ApH [2], and later with an artificially generated membrane potential difference, As [3-51. Another direction of research was addressed to structural aspects, i.e., purification and biochemical characterization of the chloroplast ATPase activities, of the CFt-part (for review see [6]) and of the CFo-Ft complex [7,8]. step should be ATP formation catalyzed by the reconstituted ATPase when energized with an artificially generated ApH or A*. This has been done by energizing CFo-Ft proteoliposomes by ApH [8] and by energizing macroliposomes reconstituted with TFo-Ft from the thermophilic bacterium PS 3 byA*[lO].

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits☆

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (KD(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The KD(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The KD(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with KD = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 104 M−1s−1. (6) The rate constant for 1-naphthoyl-ATP binding to TF1 is 6.6 · 103 M−1 · s−1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (kA−1 = 3 · 10−3s−1) and a slower second phase (kA−2 < 0.2 · 10−3s−1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.

Novel approaches towards characterization of the high-affinity nucleotide binding sites on mitochondrial F1-ATPase by the fluorescence probes 3′-O-(1-naphthoyl)adenosine di- and triphosphate

The fluorescence properties of 3-O-(1-naphthoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial F1-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and ground-state quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg2+ 120 nM; N-ATP/Mg2+ 160 nM; N-ADP/EDTA 560 nM; N-ATP/EDTA 3500 nM. For bound ligand the environment was found to be rather unipolar; the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg2+, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg2+). After the first fast step (kon greater than 1 X 10(6) M-1 X s-1), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.

Subunit-Subunit-Interactions in F0F1 as Revealed by Ligand Binding and Intrinsic Fluorescence

For a better understanding of the molecular mechanism of the H+-ATP-synthase, a prerequisite is to know more about the correlation between structural and functional aspects. One possibility to study this correlation is to compare the properties of the subunits of this enzyme complex with the changes that occur upon reconstitution of the bigger complexes. Because of its extreme stability, the ATP-synthase of the thermophilic bacterium PS3 is especially well suited for these kinds of experiments (1, 2). Using this enzyme we have compared structural properties, ligand binding and ATP hydrolysis of the holoenzyme with those of the isolated subunits. For experiments concerning ligand binding and functional properties, the adenine-nucleotide analogue naphthoyl-ATP (N-ATP) has been used (3, 4); the advantages of this ligand are: (1) being fluorescent, (2) having a high affinity for the ATP-binding sites, (3) affecting the intrinsic fluorescence of TF1, (4) being a potent inhibitor of the ATPase reaction. Preparation procedures and the methods have been described in detail elsewhere (5–7).

ATP Synthesis Catalyzed by Reconstituted CFoFl Liposomes Driven by an Artificially Generated Δψ and ΔpH

The mechanism of coupling between proton transport and ATP synthesis is still not known. One approach to study this mechanism is to simplify the energy transducing apparatus of biological menbranes and to use biochemically well-defined systems for analysis. The chloroplast ATPase CFoF1 can be isolated, purified and reconstituted into liposomes (Pick and Racker, 1979). With such a system the kinetic analysis of proton transport coupled ATP synthesis is possible.