Reconstitution reaction centers

FTIR STUDIES OF LIGHT-INDUCED INTRAMOLECULAR PROCESSES ON CRYSTALS AND RECONSTITUTED REACTION CENTERS FROM... 

The transient optical spectroscopic data show that the borohydride-treated, spheroidene-reconstituted reaction center sample is less able to form carotenoid triplet states than the native Rb. sphaeroides R26 reaction centers that have been reconstituted with spheroidene to the same extent. However, before these results can be attributed to an involvement of the bacteriochlorophyll monomer in the triplet energy transfer process, it is necessary to provide compelling evidence that spheroidene is bound in a single site, in the same environment and with the same structure in both borohydride-treated and native Rb. sphaeroides R26 reaction center samples. This evidence is provided by the following arguments (1) The absorption spectral features shown in Figs. 1 and 2 for the carotenoid in borohydride-treated and untreated, spheroidene-reconstituted complexes are very similar to each other and very different from the carotenoid in either Triton X-100 detergent or pentane. (See Fig. 3.) (2)... 

The crystallization [1] and subsequent x-ray diffraction analysis [2,3] of the reaction center from Rps, viridis have provided a detailed picture of the chromophores in their ground states, PIQaQb The crystals exhibit linear dichroism [4,5] and have been shown to be photoactive [4], A series of experiments was therefore undertaken to investigate various charge-separated states stabilized by continuous illumination (1) difference Fourier analysis of x-ray diffraction data sets for illuminated and dark crystals was performed for reaction centers reconstituted with ubiquinone-9, yielding differences between P QaQb and PQaQb/ and (2) light-induced FTIR difference spectra were measured for crystals and for reaction centers reconstituted into lipid vesicles to identify differences between Qa and Qb. Furthermore, using reconstituted reaction center samples it was possible to selectively stabilize P Qa P Qb"/ PI / PI Qa / and PI Qa. Thus the electron transfer pathway could be followed from the primary donor, P, to the secondary acceptor, Qb, via the intermediary (bacteriopheophytin) acceptor, I, and Qa. 

FTIR difference spectroscopy on reconstituted reaction centers Reaction centers were prepared as for crystallization experiments, except that the LDAO was exchanged for 0.7% CHAPS. Reaction centers were incorporated into soy bean phosphotidylcholine vesicles [15] with a final lipid concentration of 10 mg/ml and a reaction center concentration of 34 )iM. and Qb preparations were as for... 

P Qt and P Or" difference spectra. A comparison of P Qa and P Qb difference spectra for reconstituted reaction centers [8] revealed no... 

The time-resolved absorption measurements were performed on Q -reconstituted reaction centers. The data obtained at the probing wavelength of 665 nm reveal the consequences of the mutations for the binding of the quinone and for the electron transfer kinetics in the reaction centers. 

Pachence JM, Dutton PL and Blasie JK (1979) Structural studies on reconstituted reaction center-phosphatidylcholine membranes. BBA 548, 348-373. Schonfeld M, Montal M and Feher G (1979) Functional reconstitution of photosynthetic reaction centers in planar lipid bilayers, Proc.Natl.Acad. 

Near IR spectra—See Electronic absorption spectra New IR spectra, R sphaeroides reaction centers, 207,208f NH-tautomer structure, porphyrins with nonsymmetrical substitutions, 84 NH-tautomerism, porphyrins with nonsymmetrical substitution, 74-93,89-91 Ni(II)-reconstituted hemoglobin—See Nickel-reconstituted heme proteins Nickel isobacteriochlorin... 

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

The oxidative mechanism of RNR R2 differs from that of MMOH in requiring an additional electron, since Tyrl22 provides only a single electron. This electron is needed to convert P to X. It has been shown that external reductants such as excess Fe(II) or ascorbate can provide this electron in in vitro reconstitution reactions [87,97], Since the diiron site is buried 10 A below the protein surface, a long-range electron transfer pathway is required to deliver this extra electron to the diiron center. Such a pathway involving a number of amino acid residues has been proposed from examining the crystal structure of R2 [98],... 

Primary steps of photoinduced electron transfer have been studied in plant reaction centers (PS-I and PS-II), by flash absorption and EPR. In PS-I two questions wereinvestigated i) the properties of the primary radical pair P-700+, A0 (kinetics of decay nature of A0, presumably a specialized chlorophyll a decay by recombination to populate the P-700 triplet state) and ii) the nature of the secondary acceptor A,. Extraction-reconstitution experiments indicate that A, is very probably a molecule of vitamin K,. 

Heathcote, P. and Clayton, R.K. 1977. Reconstituted energy transfer from antenna pigment-protein to reaction centers isolated from Rhodopseudomonas sphaeroides. Biochim. Biophys. Acta, 459. 506-515. 

Kaufmann, K. J., Petty, K. M., Dutton, P. L., and Rentzepis, P. M., 1976, Picosecond kinetics in reaction centers of Rps. sphaeroides and the effects of ubiquinone extraction and reconstitution. Biochem. Biophys. Res. Commun., 70 839n845. 

Reconstitution of membranes from a small number of molecular components provides simplified structures to study. Thus, cytochrome oxidase or photosynthetic reaction centers, both electron transfer proteins, may be extracted from their native membranes, purified, and reincorporated at relatively high concentration into a simple well defined lipid bilayer. Diffraction investigation then provides information about the distribution and structure of the protein in the membrane. Understanding the mechanism for electron transport in these proteins will require considerable additional information. One key element of structural informations is the location of the redox centres in the membrane profile. 

As mentioned earlier, a structural model that many workers over the years had suggested for the bacterial photosynthetic apparatus consisted of the reaction center inside a cylinder of the core antenna. Based on the cylindrical structures ofLH2 ofRp. acidophila and LHl ofRs. rubrum, Kiihlbrandt presented a model in 19% for the bacterial photosynthetic unit as shown in Fig. 12 (A). It is of interest to note that in 1997 Walz and Ghosh prepared two-dimensional crystals ofthe LH1 RC complex from Rs. rubrum and obtained electron micrographs which confirmed that the RC is located inside the LHl cylinder, as LHl in the undissociated RC LHl complex has the same ring diameter as that for the reconstituted LHl reported by Karrasch et al . ... 

The availability of a photochemically active bacterial reaction-center complex has proved to be extremely useful for extraction and reconstitution experiments, the results of which can provide more conclusive evidence as to the identity of the stable primary electron acceptor in Rb. sphaeroides. 

Photochemical activity in the ubiquinone-depleted reaction centers may also be restored by readdition of other types of quinones. Reaction centers reconstituted with quinones other than ubiquinone have a different recombination times between P870 and Q in the dark, and also a different spectral widths for the broad EPR signal. Such differences are reasonable, as these parameters are expected to be sensitive to the nature of the acceptor quinone molecule. 

Removal of iron decreases the electron-transferrate from Qa to Qb by a factor of 2-3 while reconstitution with Fe, or any other metal ion investigated, restores the transfer rate. The effect of iron removal is apparently related to electrostatics rather than to the details of the electronic structure of the metal ions. Unlike Qa in a native reaction center, Qa in Ee-depleted reaction centers can become a two-electron acceptor, indicating that Qa is accessible to solvent protons when iron is absent. Even though these results indicate that iron does not play an obligatory role in electron transfer from Qa to Qb, some other possible functional or even structural role of iron in photosynthetic organisms may yet be uncovered. 

In 1970, Dan Reed, Tom Chaney and this author used a cytochrome-free, reaction-center complex from Rb. sphaeroides R-26 and tried to reconstitute it with mammalian cytochrome c in an attempt to mimic in vivo electron transfer. Although P870 can undergo rapid oxidation by a light flash, its rereduction is very slow, as expected, in the absence of efficient secondary donors, as shown in Fig. 12, upper row. However, P870 may be reduced very rapidly by an externally added redox mediator such as reduced PMS. For example, in the presence of 0.1 mM reduced PMS, P870 can be re-reduced in 36 /js. 

Ki Satoh, Hansson and P Mathis (1990) Charge recombination between stabilized P680 and reduced cytochrome b559 in quinone-reconstituted PS II reaction center. Biochim Biophys Acta 1016 121-126... 

In their earlier work, Michel, Epp, and Deisenhofer crystallized Rp. viridis reaction center with the vacant Qg site reconstituted with either o-phenanthroline (OP) or terbutryn (2-thiomethyl-4-ethylamino-6-t-butylamino-5 -triazine and abbreviated as TERB ) and were able to identify three binding locations for OP and one for TERB. In Eig. 10 (B), OP is shown seated deep within the pocket. Each of the two N-atoms (shaded) of OP form a hydrogen bond with the imidazole nitrogen of His 190. In addition, OP is also close to Ile229 and Leu 193 (neither shown in the figure). 

DJ Chapman, I Vass and J Barber (1991) Secondary electron transfer reactions of isolated photosystem II reaction center after reconstitution with plastoquinone-9 and diacylglycerolipids. Biochim Biophys Acta 1057 391-398... 

As mentioned above, a 1 1 stoichiometric relationship between the photooxidized donor, P700 and the reduced terminal acceptors has not yet been established for the PS-I reaction center. As previously noted, the total amount of the recognized terminal acceptors reduced at 15 K is, on average, approximately 74% of the P700 photooxidized. Even more intriguing, in the reconstituted PS-I complexes from either the Cys-14->Asp or Cys-51->Asp mutant PsaC protein, the extent of photoreduction of each intact iron-sulfur cluster at 15 K remained nearly the same as in the wild-type preparation. The presence of the other cluster that was made photochemically inactive by site-directed mutagenesis apparently had no effect on the behavior of the unaltered cluster. 

As noted above, PsaC, i.e., FeS-A/FeS-B, can be removed from the native PS-1 reaction-center complex by treatment with chaotropic agents. However, it was in 1988 that Golbeck, Mehari, Parrett and Ikegami succeeded in reconstituting the isolated PS-1 core protein with isolated PsaC and obtained a... 

Fig. 8. (A) Schematic representation of reconstitution of the PS-i core complex [P700 FeS-X] with PsaC (B) Light-induced absorbance change (LI-AA) at 698 nm in the PS-1 core complex and in the reconstituted complex (C) EPR spectra of the core complex, PsaC and the reconstituted complex. Panel (D) is the EPR spectrum of the native PS-1 reaction-center complex. See text for experimentai details. Figure source Golbeck, Mehari, Parrett and Ikegami (1988) Reconstitution of the photosystem i compiex fmm the P700 and Fx-containing reaction center core protein and the FfjF polypeptide. FEBS Lett 240 10, 11, 12.

JH Golbeck, T Meharl, K Parrett and I Ikegami (1988) Reconstitution of the photosystem 1 complex from the P700 and Fx-containing reaction center core protein and the F /Fg polypeptide. FEBS Lett 240 9-14... 

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