The incorporation of reaction centres into membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides

Reaction centres purified from a blue-green mutant R-26 of Rhodopseudomonas sphaeroides can be incorporated into bacteriochlorophyll-less membranes purified from an aerobically-grown bacteriochlorophyll-less mutant 01 of R. sphaeroides. This can be accomplished by raising the temperature of the mixture or by addition of the detergent sodium cholate and its subsequent removal by dilution or dialysis. Optimum conditions for the reconstitution are at 4°C in the presence of 1% cholate and soybean phospholipid (2 : 1, w/w, with membrane protein). Isopycnic sucrose density gradient centrifugation of such preparations shows that reaction centres and light-harvesting pigment-protein complex bind to the membranes. Reconstituted membranes exhibit light-induced steady-state cytochrome absorbance changes resembling those observed in chromatophores prepared from the photosynthetically-grown mutant R-26. The effect on these absorbance changes of varying reaction centre content in the membrane has been studied, and the time course of the interaction between 01 membrane cytochrome c₂ and added reaction centre examined.Cytochrome b photoreduction and cytochrome c₂ photo-oxidation were observed in the reconstituted preparation; each increased following the addition of antimycin A, suggesting that a cyclic light-driven system had been reconstituted.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes.

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dislysis, the dislysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c2 photooxidation and cytochrome b photoreduction was demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane. The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides. Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c2 photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greated membrane cytochrome c2 photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.     

Crystallographic studies of mutant reaction centres from Rhodobacter sphaeroides

X-ray structures have been determined for five mutant reaction centres from Rhodobacter sphaeroides, at resolutions varying between 3.4 Å and 2.3 Å. The aim was to examine the effects of mutagenesis of polar residues in the binding pocket of the reaction centre carotenoid. The number of water molecules identified in each structure depended on the resolution and completeness of the data. In a 2.3 Å structure for a WM115F/FM197R mutant reaction centre, two water molecules partially filled the cavity that was created when the tryptophan residue was replaced by a less bulky phenylalanine. Structures obtained for four reaction centres with mutations of polar residues in the carotenoid binding pocket failed to show any significant change in the structure of the reaction centre carotenoid. Low resolution data for a YM210W mutant reaction centre showed that the overall structure of this complex is well conserved. This finding is discussed in light of the intriguing spectroscopic properties of the YM210W mutant reaction centre, and an alternative pathway for transmembrane electron transfer identified in this mutant.     

Reconstituted energy transfer from antenna pigment-protein to reaction centres isolated from Rhodopseudomonas sphaeroides.

Efficient energy transfer has been reconstituted between an antenna pigment-protein and reaction centres isolated from the photosynthetic membrane of Rhodopseudomonas sphaeroides. The reconstituted system has fluorescence induction kinetics and fluorescence yields similar to those obtained from antenna bacteriochlorophyll in chromatophores. The results indicated that closed reaction centres quench fluorescence from the antenna pigment-protein, although not as strongly as photochemically active reaction centres. The measurement of fluorescence yields from chromatophores of the reaction centreless mutant PM-8 and of the parent strain Ga confirmed these observations. The fluorescence yield from the reconstituted system was approximately the same whether the reaction centres had been closed by photo-oxidation of the bacteriochlorophyll electron donor or chemical reduction of the primary acceptor, indicating a similar lifetime for the excited singlet state in both states of the reaction centres.     

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomonas sphaeroides.

The bacterial reaction center absorbance change at 450 nm (A-450) assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the "true" primary acceptor is reduced. Alternatively, the primary acceptor may act in a "ping-pong" fashion with respect to subsequent photoelectrons.     

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides.

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll a . protein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

The kinetics of flash-induced electron flow in bacteriochlorophyll-less membranes of Rhodopseudomonas sphaeroides reconstituted with reaction centres

Membranes isolated from aerobically grown mutants 01 and PM8bg II-15 of Rhodopseudomonas sphaeroides lack reaction centres. Incorporation of purified reaction centres into these membranes can be achieved by mixing the protein and membranes in 1% sodium cholate with added soybean phospholipid and removing the cholate by dialysis.The kinetics of light-stimulated electron flow in these reconstituted membranes have been examined and compared with those observed in chromatophore membranes isolated from photosynthetically grown R. sphaeroides. Following a single saturating flash, reconstituted reaction centres become photo-oxidised, and about 60% are re-reduced within about 200 ms by cytochrome c2 in the 01 membrane. Cytochrome c2 photo-oxidation is biphasic, the half-time of the first fase being faster than 20 μs. The second phase is variable and can be as slow as 60 ms. A cytochrome b in the membrane becomes photoreduced with a half-time of 27 ms. Electron flow between cytochromes b and c2 is slow and appears only partially sensitive to antimycin A.Using membranes from the reaction centre-less mutant PM8bg II-15 similar reconstitution measurements were performed. The resulting kinetic measurements showed that fast cytochrome b photoreduction and cytochrome c2 photo-oxidation occurred.The absorbance change at 560 minus 570 nm induced by steady-state illumination of 01 membranes reconstituted with reaction centres was measured at a range of ambient potentials; the reaction was abolished at oxidation-reduction potentials below 0 mV. The change was approximately halved at +50 mV, indicating that cytochrome b₊₅₀ is the recipient of electrons from the reconstituted reaction centres.     

Hydrogen formation in nearly stoichiometric amounts from glucose by a Rhodopseudomonas sphaeroides mutant.

Rhodopseudomonas sphaeroides produces molecular H2 and CO2 from reduced organic compounds which serve as electron sources and from light which provides energy in the form of adenosine 5'-triphosphate. This process is mediated by a nitrogenase enzyme. A mutant has been found that, unlike the wild type, will quantitatively convert glucose to H2 and CO2. Techniques for isolating other strains capable of utilizing other unusual electron sources are presented. Metabolism of glucose by the wild-type strain leads to an accumulation of gluconate. The isolated mutant strain does not appear to accumulate gluconate.     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

Protein fluorescence of photosynthetic reaction centers from Rhodopseudomonas sphaeroides

Luminescence emitted by tryptophan residues of reaction center (RC) preparations was studied. The RG preparations were isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides by treatment with lauryl dimethyl amine oxide (LDAO). After excitation at lambda 280 nm the quantum yield of luminescence is 0,02. It is shown that 60% of tryptophanyls are located inside the protein globule in the surrounding of relaxating polar groups and the rest approximately 40% on the outer surface of the globule--predominantly in the positively charged region of the LDAO-RC protein--in the surrounding of protein-bound water molecules. There is a correlation between the pH dependencies of the position of the peak of luminescence from tryptophanyls and effectivity of electron transfer from the primary (quinone) to secondary acceptor. The two parameters are invariant at pH from 7 to 9 and vary at pH less than 7 and pH greater than 9. The phenomena responsible for the observed correlation are discussed on the basis of pH-dependent changes in the RC protein which govern electron transport activity at the reaction center.     

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.     

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.     

Transmembrane orientation of reaction centers in proteoliposomes from Rhodopseudomonas sphaeroides

The photochemical reaction centers from Rhodopseudomonas sphaeroides were reconstituted with soybean phospholipids into liposomes by the cholate-dialysis method. The transmembrane orientation of the reaction centers in the proteoliposomes and the morphology of the vesicles were investigated. The orientation was determined by the reduction of externally added cytochrome c after its photooxidation by a flash. The structure of the vesicles was examined by electron microscope. Discontinuous sucrose density gradient centrifugation yielded several proteoliposome fractions with different vesicular sizes and reaction-center orientations. The proportion of the reaction centers that exposed their cytochrome c reacting sites to the outside of the vesicles increased from 45 to 85% with an increase of the vesicular size. The proportion also depended on the ionic composition of the dialysis buffer. The optimal ionic environment during the dialysis (100 mM NaCl or 2.5 mM MgSO4) gave a liposome yield of 25-30% with a highly asymmetric orientation (greater than 60%). Entrapping of cytochrome c molecules into the phospholipid vesicles had little effect on the orientation of the reaction centers.     

Rhodopseudomonas sphaeroides forma sp. denitrificans,a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.