Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of 14 subunits arranged with 72 point group symmetry, and oligomeric (L8S8) Rubisco was obtained.     

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Supercomplexes of IsiA and photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Structure and subunit arrangement of the A-type ATP synthase complex from the archaeon Methanococcus jannaschii visualized by electron microscopy

In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei G?1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases.     

Characterization of a redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor flavodoxin.

A covalent complex between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was generated by chemical cross-linking. Laser flash-absorption spectroscopy indicates that the bound flavodoxin of this complex is stabilized in the semiquinone state and is photoreduced to the quinol form upon light excitation. The kinetics of this photoreduction process, which takes place in approximately 50% of the reaction centres, displays three exponential components with half-lives of 9 microsec, 70 microsec and 1 ms. The fully reduced flavodoxin subsequently recombines with P700+ with a t1/2 of 330 ms. A corresponding flavodoxin semiquinone radical signal is readily observed in the dark by room temperature electron paramagnetic resonance, which reversibly disappears upon illumination. In contrast, the light-induced reduction of oxidized flavodoxin can be observed only by first-flash experiments following excessive dark adaptation. In addition, the docking site of flavodoxin on photosystem I was determined by electron microscopy in combination with image analysis. Flavodoxin binds to the cytoplasmic side of photosystem I at a distance of 7 nm from the centre of the trimer and in close contact to a ridge formed by the subunits PsaC, PsaD and PsaE.     

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.