RC Complexes Research Articles

Isotopic labelling of the polypeptide subunits of the isolated photosystem II reaction-centre complex of Chlamydomonas reinhardtii suggests an αβ heterodimeric structure for cytochrome b-559

The photosystem II reaction-centre (PSII RC) complex contains five polypeptides: D1, D2, PsbI and the α and β subunits of cytochrome b-559. In order to determine their stoichiometry, we have isolated a PSII RC from a mutant of the green alga Chlamydomonas reinhardtii, lacking the photosystem I and chloroplast ATP-ase complexes, which has been grown in the presence of [ 14C]-acetate. The stoichiometry of the polypeptides is estimated by SDS-polyacrylamide electrophoresis followed by phosphorimage analysis of the gel. Our data suggest equimolar amounts of each polypeptide. Assuming the presence of homogeneous PSII RC complexes, our results are consistent with an αβ heterodimeric structure for cytochrome b-559.

A binary complex of the catalytic subunit of cAMP-dependent protein kinase and adenosine further defines conformational flexibility.

cAMP-dependent protein kinase (cAPK), a ubiquitous protein in eukaryotic cells, is one of the simplest members of the protein kinase family. It was the first protein kinase to be crystallized and continues to serve as a biochemical and structural prototype for this family of enzymes. To further understand the conformational changes that occur in different liganded and unliganded states of cAPK, the catalytic subunit of cAPK was crystallized in the absence of peptide inhibitor. The crystal structure of the catalytic subunit of mouse recombinant cAPK (rC) complexed with adenosine was solved at 2.6 A resolution and refined to a crystallographic R factor of 21.9% with good stereochemical parameters. This is the first structure of the rC subunit that lacks a bound inhibitor or substrate peptide. The structure was solved by molecular replacement and comprises two lobes (large and small) which contain a number of conserved loops. The binary complex of rC and adenosine adopts an 'intermediate' conformation relative to the previously described 'closed' and 'open' conformations of other rC complexes. Based on a comparison of these structures, the induced fit that is necessary for catalysis and closing of the active-site cleft appears to be confined to the small lobe, as in the absence of the peptide the conformation of the large lobe, including the peptide-docking surface, does not change. Three specific components contribute to the closing of the cleft: rotation of the small lobe; movement of the C-terminal tail; and closing of the so-called glycine-rich loop. There is no induced fit in the large lobe to accommodate the peptide and the closing of the cleft. A portion of the C-terminal tail, residues 315-334, serves as a gate for the entry or exit of the nucleotide into the hydrophobic active-site cleft.

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu(2+) ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO4 and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (ΔA of 430-440 nm) and by the flash induced formation of P680(+) (ΔA at 820 nm). The relatively slow relaxation kinetics of the latter signal (t1/2 ≈ 1 ms) may suggest that in a substantial fraction of the RCs QA remains bound to the complex.

Characterization of reaction center/antenna complexes from bacteriochlorophyll a containing Ectothiorhodospiraceae

Pigment-protein complexes have been isolated from three bacteriochlorophyll- a -containing Ectothiorhodospira species: E. mobilis, E. shaposhnikovii and E. halophila , using detergent solubilisation of photosynthetic membranes and sucrose-density gradient centrifugation. This isolation procedure yields pure LHII complexes and reaction centers with either one or both antenna complexes attached (i.e., RC/LHI and RC/LHI/LHII complexes). The RC/LHI/LHII complexes consist of three reaction center polypeptides, H (35 kDa), M (34 kDa), and L (26.5 kDa), plus two LHI and two LHII antenna polypeptides. In photosynthetic membranes of the three Ectothiorhodospira species two major types of heme c are present, of low (25 mV) and high (297 mV) midpoint potential, respectively. These heme groups belong to separate cytochromes. During solubilisation and purification, no loss of the H subunit from the reaction centers is observed. However, the reaction centers do lose their c -type cytochromes. The high-potential cytochrome c is completely lost during solubilisation of RC complexes from photosynthetic membranes, while the low-potential cytochrome is largely lost during isolation of RC/LHI complexes. The residual amount of this low-potential cytochrome, associated with the RC/LHI complexes, varies with the salt concentration used during the isolation, but remains below 0.2 mol heme c per mol reaction center. This is less than 20% of the amount originally present in photosynthetic membranes.

Low-temperature photooxidation of cytochrome c in reaction centre complexes from Rhodopseudomonas viridis

Low-temperature photooxidation of the high-potential haem c 380 ( c-559) and low-potential haem c 20 ( c-552) has been studied in isolated RC complexes from Rps. viridis. At appropriately low redox potential, complete irreversible oxidation of c 20 ensues following flash-excitation at 77 K with t 1 2 = 20 μ s . Photooxidation of c 380, which can be observed at high E h, is more rapid ( t 1 2 < 6 μ s ) and fully reversible; however, only about 20% of the haem is oxidized at 77–140 K. Full photooxidizability of c 380 is attained as the temperature is raised in the range 140–200 K. Studies of the charge recombination kinetics indicate the low temperature-induced decrease of c 380 photooxidizability to originate from the effect of freezing on the equilibrium constant of electron distribution between the haem and the pigment: ▪ K eq decreasing from about 100 at 293 K to about 0.25 below 140 K. A two-step mechanism of c 380 photooxidation above 200 K is suggested where initial virtually isopotential temperature-independent electron dislocation from c 380 to P is followed by nuclear medium reorganization which stabilizes the final c + 380 P state: ▪ Freezing is likely to prevent reorganization of the medium, thus allowing for observation of the initial electron transfer step at low temperatures. Photooxidation of the low-potential haem(s) may occur via the equilibrium fraction of the c + 380 P state with unrelaxed nuclear environment. In sum, the results of low-temperature experiments are fully consistent with the linear sequence of haems ▪ and there is no need to postulate parallel electron transfer pathways involved in photooxidation of high- and low-potential haems.

Orientation of reaction center complexes fromRhodobacter sphaeroides in proteoliposomes and the effect ofo-phenanthroline on electrogenesis during primary photochemical reaction

The orientation of Rhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown that o-phenanthroline, an inhibitor of electron transfer between primary (QA) and secondary (QB) quinone acceptors, can also inhibit the photoinduced QA reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-induced o-phenanthroline inhibition of primary dipole (P-870+.QA-) formation is of a competitive nature.

Effects of histrionicotoxin derivatives on ion channels and acetylcholine receptor-channel complexes in bullfrog sympathetic ganglia

1. Four synthetic histrionicotoxin derivatives (H8-, H12-, C4H10- and C5H10-HTX) were applied to bullfrog (Rana catesbeiana) sympathetic ganglia and their effects were compared electrophysiologically. 2. The derivatives (60-100 microM) blocked both acetylcholine receptor-channel complex (ACh RC complex) and Na+ channel to cause a transmission failure. They also blocked K+ and Ca2+ channels. 3. Although all 4 derivatives exhibited similar effects, their potencies on respective ionic channels differed from one another. 4. Two types of (presumably subsynaptic and extrasynaptic) ACh RC complexes in ganglion cells were distinguished based on their differential sensitivities to HTX derivatives.