Wild-type Reaction Centers Research Articles

Primary Electron Transfer in Membrane-Bound Reaction Centers with Mutations at the M210 Position

The kinetics of primary electron transfer in membrane-bound Rhodobacter sphaeroides reaction centers (RCs) were measured on both wild-type (WT) and site-directed mutant RC's bearing mutations at the tyrosine M210 position. The tyrosine was replaced by histidine (H), phenylalanine (F), leucine (L), or tryptophan (W). A mutant with histidine at both the M210 and symmetry-related L181 positions (YM210H/FL181H) was also examined. Rates of primary charge separation were determined by both single and multiple wavelength pump−probe techniques. The time constants for the decay of stimulated emission in the membrane-bound mutant RC's were approximately 27 ps (F), 36 ps (L), 72 ps (W), 5.8 ps (H), and 4.2 ps (HH), compared with 4.6 ps in WT membrane-bound RC's. For all RC's, the decay of stimulated emission was found to be multiexponential, demonstrating that this phenomenon is not a consequence of the removal of the RC from the membrane. The source of the multiexponential decay of the primary donor excited state w...

Distant electrostatic interactions modulate the free energy level of QA- in the photosynthetic reaction center.

In the reaction centers from the purple photosynthetic bacterium Rhodobacter capsulatus, we have determined that residue L212Glu, situated near the secondary quinone acceptor QB, modulates the free energy level of the reduced primary quinone molecule QA- at high pH. Even though the distance between L212Glu and QA is 17 A, our results indicate an apparent interaction energy between them of 30 +/- 18 meV. This interaction was measured by quantitating the stoichiometry of partial proton uptake upon formation of QA- as a function of pH in four mutant strains which lack L212Glu, in comparison with the wild type. These strains are the photosynthetically incompetent site-specific mutants L212Glu -->Gln and L212Glu-L213Asp-->Ala-Ala and the photocompetent strains L212Glu-->Ala and L212Ala-L213Ala-M43Asn-->Ala-Ala-Asp. Below pH 7.5, the stoichiometry of proton uptake from all strains is nearly superimposable with that of the wild type. However, at variance with the wild type, reaction centers from all strains that lack L212Glu fail to take up protons above pH 9. The lack of a change in the free energy level of QA- at high pH in the L212Glu-modified strains is confirmed by the determination of the pH dependence of the rate (kAP) of P+QA- charge recombination in the reaction centers where the native QA is replaced by quinones having low redox potentials. Contrary to the wild-type reaction centers where kAP increases at high pH, almost no pH dependence could be detected in the strains that lack L212Glu. Our data show that the ionization state of L212Glu, either on its own or via interactions with closely associated ionizable groups, is mainly involved in the proton uptake at high pH by reaction centers in the PQA- state. This suggests that the formation of the QA- semiquinone state induces shifts in pKas of residues in the QB proteic environment. This long-distance influence of ionization states is a mechanism which would facilitate electron transfer from QA to QB on the first and second flashes. The functional communication between the two quinone protein pockets may involve the iron-ligand complex which spans the distance between them.

Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier.

We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide. Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding. In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL. The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs. This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine. In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta). This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle. Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type. The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.

Kinetics and free energy gaps of electron-transfer reactions in Rhodobacter sphaeroides reaction centers.

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)210 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll dimer (P*) transfers an electron to a bacteriopheophytin (HL) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- 2, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from HL- to a quinone (QA) with a time constant of 0.16 ns in wild-type RCs, 0.24 ns in the Phe mutant, and 0.20 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 pcs in the Trp mutant. Most of the electron transfer to HL occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (Em) of the P/P+ redox couple is measured by an electrochemical technique; the Em values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 552 +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (delta G degrees) associated with formation of the P+HL-radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from HL- to QA. The free energy of P+HL- is elevated by an amount comparable to that calculated from the increase in the Em of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 2. Quantitative kinetic analysis

The electron-transfer kinetics from the soluble cytochrome (cyt) c2 to the photooxidized reaction center (RC) was studied with proteins isolated from Rhodobacter (R.) sphaeroides. In addition to wild-type (WT) RC, RCs harboring site-directed mutations at residue L162 (L162F, -M, -L, -S, or -G) wree analyzed. The disappearance of the absorption band of the photooxidized primary donor P+ (at 1250 nm) and the alpha-band of cyt c2 (at 550 nm) were monitored. Under conditions of high equimolar RC and cyt c2 concentrations, the kinetics were very similar to those measured in intact cells (Farchaus et al., 1993). The fast component of the kinetics normally seen in WT was not observed in any of the mutants; the overall rereduction rates for the mutants depended on the amino acid substitution. Light intensity, viscosity, ionic strength, and RC/cyt c2 stoichiometry of the reaction mixture were varied to distinguish the contributions of association, reorientation, and electron-transfer reactions to the observed kinetics. In competition experiments, WTRC (L162Y) and the mutant RCL162L showed similar affinity for cyt c2, with a dissociation constant of kD = 10(-6) M. Mutants with an aliphatic substitution at position L162 displayed slower cyt c2-RC association and dissociation rates. Comparison of the major kinetic component of the P+ rereduction rates for the aliphatic substitutions to the aromatic substitution, L162F, revealed that the former were less affected by ionic strength and viscosity than the latter. The viscosity and ionic strength dependences noted for L162F were comparable to those seen for the slow kinetic component observed for the WT RC. The redox midpoint potential of the P/P+ couple was increased by 30 mV (L162F) to 50 mV (L162L, G) over the WT value, leading to differences in delta G not large enough to account for the drastic kinetic effects. Rather, the results suggested that the state(s) where cyt c2 is nonproductively bound to the RC dominated in the mutants. In the L162F mutant, it appeared that only the distribution between the bound cyt c2 states was affected, whereas for the mutants with aliphatic substitutions, a decreased reorientation rate had to be additionally assumed in order to explain the observations.

Triplet state EPR of reaction centers from the HisL173?LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His(L173)→Leu(L173) substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (∼90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu(L173) mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu(L173) mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His(M200)→Leu(M200) mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.

Subpicosecond emission studies of bacterial reaction centers

The spontaneous emission of reaction centers from native and mutated Rhodobacter sphaeroides and from wild type Chloroflexus aurantiacus is investigated by fluorescence up-conversion with high temporal resolution. The time constant of 0.9 ps previously observed in transient absorption experiments on wild type reaction centers of Rhodobacter sphaeroides does not appear in the emission experiment. However, all investigated reaction centers display a biexponential decay of the emission with time constants in the 2 ps to 25 ps range. The experimental results are discussed within the frame of different reaction models including a possible sample heterogeneity or a transient electron transfer to the inactive pigment branch.

Photochemical trapping of a bacteriopheophytin anion in site-specific reaction-center mutants from the photosynthetic bacterium Rhodobacter sphaeroides.

The mutant YY in the reaction center of Rhodobacter sphaeroides, in which Phe181 on the L chain has been replaced by Tyr, and the double mutant FY, with Tyr210 on the M chain replaced by Phe and Phe181 on the L chain replaced by Tyr, have been constructed by site-directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photoheterotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I-) indicates that the bacteriopheophytin on the B branch is reduced to a much greater extent in FF and FY as compared to YY and wild-type YF. Low temperature (77 K) absorption spectra clearly show that in the wild-type (YF) and YY reaction centers only the 545-nm-absorbing bacteriopheophytin is reduced while in the FF and FY reaction centers both the 535-nm and 545-nm-absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to explain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the 'cycling' time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward electron transfer rate.

Structure of the detergent phase and protein-detergent interactions in crystals of the wild-type (strain Y) Rhodobacter sphaeroides photochemical reaction center

Rhodobacter sphaeroides (strain Y) reaction center (RC) crystals were grown in the presence of n-octyl beta-glucoside (beta-OG). In order to determine the structure of the detergent phase in these crystals, low-resolution neutron diffraction experiments were performed at different contrasts obtained by varying the H2O/D2O ratio in the solvent. From the contrast variation data and from the RC atomic coordinates determined by X-ray diffraction [Arnoux, B., Ducruix, A., Reiss-Husson, F., Lutz, M., Norris, J., Schiffer, M., & Chang, C. H. (1989) FEBS Lett. 258, 47-50], a model was obtained for the structure of the detergent phase in the crystal. The detergent forms a ring-shaped micelle surrounding the most hydrophobic part of the transmembrane alpha helices of the RC. Each detergent ring is connected to two next-neighbor rings by intermicellar bridges. The detergent phase is organized thus in infinite zigzag chains parallel to the b axis of the P2(1)2(1)2(1) unit cell. The main interactions between beta-OG molecules and the RC molecules are hydrophobic and are localized at the level of the transmembrane alpha helices. This interaction replaces the phospholipid-protein interaction existing in vivo in the membrane and, to some extent, also the light harvesting complex-protein interaction. Secondary hydrophilic interactions are found between a few of the charged residues of the H subunit and the hydrophilic surface of the detergent ring from a neighboring RC molecule. A comparison with a previous study on Rhodopseudomonas viridis crystals [which grow in the presence of lauryldimethylamine N-oxide (LDAO) and belong to a different space group] [Roth, M., Lewit-Bentley, A., Michel, H., Deisenhofer, J., Huber, R., & Oesterhelt, D. (1989) Nature 340, 659-661] shows a quasi identity of shape and position of the beta-OG and LDAO rings around the transmembrane alpha helices. The secondary interactions, involving in both cases the external surface of the H subunit, differ because of the different molecular packing in the two space groups. The role and structural requirements of the detergent in the crystallization process are discussed.

Investigation into the source of electron transfer asymmetry in bacterial reaction centers

We have investigated the primary photochemistry of two symmetry-related mutants of Rhodobacter sphaeroides in which the histidine residues associated with the central Mg2+ ions of the two bacteriochlorophylls of the dimeric primary electron donor (His-L173 and His-M202) have been changed to leucine, affording bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimers. Reaction centers (RCs) from the two mutants, (L)H173L and (M)H202L, have remarkably similar spectral and kinetic properties, although they are quite different from those of wild-type RCs. In both mutants, as in wild-type RCs, electron transfer to BPhL and not to BPhM is observed. These results suggest that asymmetry in the charge distribution of the excited BChl dimer (P*) in wild-type RCs (due to differing contributions of the two opposing intradimer charge-transfer states) contributes only modestly to the directionality of electron transfer. The results also suggest that differential orbital overlap of the two BChls of P with the chromophores on the L and M polypeptides does not contribute substantially to preferential electron transfer to BPhL.

Charge Separation in a Reaction Center Incorporating Bacteriochlorophyll for Photoactive Bacteriopheophytin

Site-directed mutagenic replacement of M subunit Leu214 by His in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides results in incorporation of a bacteriochlorophyll molecule (BChl) in place of the native bacteriopheophytin (BPh) electron acceptor. Evidence supporting this conclusion includes the ground-state absorption spectrum of the (M)L214H mutant, pigment and metal analyses, and time-resolved optical experiments. The genetically modified RC supports transmembrane charge separation from the photoexcited BChl dimer to the primary quinone through the new BChl molecule, but with a reduced quantum yield of 60 percent (compared to 100 percent in wild-type RCs). These results have important implications for the mechanism of charge separation in the RC, and rationalize the choice of (bacterio)pheophytins as electron acceptors in a variety of photosynthetic systems.

Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.

Charge transfer and charge resonance states of the primary electron donor in wild-type and mutant bacterial reaction centers

Low-temperature subpicosecond measurements on heterodimer-containing reaction centers from the His M200 → Leu mutant of Rhodobacter capsulatus are reported. As at room temperature, primary electron transfer is initiated from a state of the bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimer with substantial [BChl +BPh −] intradimer charge transfer character. The electronic composition of this transient state is discussed in terms of mixing of the exciton and charge resonance states of the BChl/BPh dimer and compared with the nature of the excited states of the BChl/BChl dimer in wild-type reaction centers.

Towards the understanding of the function of Rb sphaeroides Y wild type reaction center: gene cloning, protein and detergent structures in the three-dimensional crystals

We report various experiments aimed at the resolution of the 3-dimensional structure of the photosynthetic reaction center from wild type Y Rhodobacter sphaeroides. The genes encoding the L and M polypeptides have been cloned and sequenced. They bear 2 mutations each when compared to those already sequenced in another Rb sphaeroides strain (2.4.1). In the L gene, these codon changes are silent. In the M gene, one is silent and the other one leads to a Leu- Met substitution at position 140. At the present stage of the refinement of the X-ray data (0.3 nm resolution) the structure of the Y reaction center is shown to be highly similar to that of the Rhodopseudomonas viridis reaction center. The binding of spheroidene on the M side of the Y reaction center is shown to be determined by hydrophobic interactions with neighboring amino acids and by steric factors. Preliminary results concerning the localization of the detergent (β-octylglucoside) in the unit cell are presented. This method combines low angle neutron scattering at different contrasts in H 2O/D 2O with X-ray crystallographic data.

Stark effect in wild-type and heterodimer-containing reaction centers from Rhodobacter capsulatus

The effect of an external electric field on the optical absorption spectra of wild-type Rhodobacter capsulatus and two Rb. capsulatus reaction centers that have been genetically modified through site-directed mutagenesis (HisM200----LeuM200 and HisM200----PheM200) was measured at 77 K. The two genetically modified reaction centers replace histidine M200, the axial ligand to the M-side bacteriochlorophyll of the special pair, with either leucine or phenylalanine. These substitutions result in the replacement of the M-side bacteriochlorophyll with bacteriopheophytin, forming a bacteriochlorophyll-bacteriopheophytin heterodimer. The magnitude of the change in dipole moment from the ground to excited state (delta mu app) and the angle delta between the Qy transition moment and the direction of delta mu app were measured for the special pair absorption band for all three reaction centers. The values for delta mu app and delta obtained for wild-type Rb. capsulatus (delta mu app = 6.7 +/- 1.0 D, delta = 38 +/- 3 degrees) were the same within experimental error as those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. The values for delta mu app and delta obtained for the red-most Stark band of both heterodimers were the same, but delta mu was substantially different from that of wild-type reaction centers (HisM200----LeuM200, delta mu app greater than or equal to 14.1 D and delta = 33 +/- 3 degrees; HisM200----PheM200, delta mu app greater than or equal to 15.7 D and delta = 31 +/- 4 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic study of PF and CarT states in the LM subunit purified from the wild-type Rhodobacter sphaeroides reaction centers

The kinetics of absorbance changes related to the charge-separated state, P(F), and to the formation and decay of the carotenoid triplet state (Car(T)) were studied in the LM reaction center subunit isolated from a wild-type strain of the purple bacterium Rhodobacter sphaeroides (strain Y). The P(F) lifetime is lengthened (20±1.5 ns) in the LM complex as compared to the intact reaction centers (11±1 ns). The yield of the carotenoid triplet formation is higher (0.28±0.01) in the LM complex than in native reaction centers. We interpret our results in terms of perturbations of a first-order reaction connecting the singlet and the triplet state of the radical-pair state. Our results, together with those of a recent work (Agalidis, I., Nuijs, A.M. and Reiss-Husson, F. (1987) Biochim. Biophys. Acta (in press)) are consistent with a high I to QA electron transfer rate in this LM subunit, which is metal-depleted.The LM complex is considerably more sensitive than the reaction centers to photooxidative damage in the presence of oxygen. This is not readily accounted for simply by the higher carotenoid triplet yield, and may suggest a greater accessibility of the internal structures in the absence of the H-subunit.The lifetime of the carotenoid triplet decay (6.4±0.3 μs) in the LM subunit is unchanged compared to the native reaction centers.

Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata

Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-harvesting II (LHII) structural genes have been deleted. In one series of strains, the 2778-bp ApaI fragment bearing more than 90% of the rxcA operon (promoter and structural genes coding for LHIβ, LHIα, RC-L and RC-M) has been deleted from the chromosome. When the rxcA operon is deleted, resultant strains possess only LHII and are photosynthetically defective. The rxcA deletion in an LHII − background results in a strain lacking all LH antennae and RC subunits. As expected this strain has no near-infrared absorption characteristic of LH or RC bacteriochlorophyll. The rxcA deletion may be complemented by a pBR322 derivative carrying the entire rxcA operon (pU21). In a second series of deletion mutants, the 2500-bp BstEII- StuI fragment, including the β and α structural genes coding for LHII has been deleted from the chromosome. In the wild-type background, functional RC and LHI are synthesized. LHII may be restored in the deletion strain by conjugal transfer of the plasmid pU2 which carries the LHII operon.

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.

Several properties of the LM unit extracted with sodium dodecyl sulfate from Rhodopseudomonas sphaeroides purified reaction centers

We describe several characteristics of an LM unit extracted from purified wild-type Rhodopseudomonas sphaeroides reaction centers after treatment with SDS and lauryldimethylamine N-oxide. We also studied another preparation called RC-SDS in which after a similar detergent treatment the H-chain was not separated from the LM unit. The spectral properties of both preparations were similar to those of intact reaction centers; the main differences were a blue shift of the P-865 Q y band and a narrowing of the bacteriopheophytin Q x band. Studies of absorbance changes after steady light or flash illumination showed that LM was depleted of the primary electron acceptor Q I and of the transition metal (Mn), but was still able to react with quinones, with a partial restoration of the photochemistry. The primary acceptor Q 10 was still partly present in RC-SDS and formed in the light a very unstable semiquinone anion. In both preparations, in the presence of added quinones, the flash-induced P-865 + decayed in the dark with multiexponential kinetics, the apparent half-times ranging between 60 ms and 100 s. This recovery was accelerated either by the absence of O 2 or (in the case of LM only) by o-phenanthroline, which also decreased the amplitude of the P-865 + signal. From these experiments it was concluded that in these preparations the site of binding of the secondary quinone Q II was damaged or absent; the altered properties of this LM unit were considered to result from the denaturing action of the detergents, rather than from the absence of the H polypeptide.

Binding of carotenoids on reaction centers from Rhodopseudomonas sphaeroides R 26

The carotenoid-less reaction centers isolated from Rhodopseudomonas sphaeroides (strain R 26) bind pure all-trans spheroidene as well as spheroidenone in a nearly 1:1 molar ratio with respect to P-870. Neither β-carotene nor spirilloxanthin, both absent from wild-type Rps. sphaeroides, could be bound in appreciable amounts. Resonance Raman spectra of the carotenoidreaction center complex indicate that the carotenoid is bound as a cis isomer, its conformation being very close, although probably not identical, to that assumed by the carotenoid in the wild-type reaction centers. The electronic absorption spectra of the carotenoid-reaction center complexes are in good agreement with such a interpretation. When bound to the R 26 reaction centers, spheroidene displays light-induced absorbance changes identical in peak wavelengths and comparable in amplitudes to those observed in the wild-type reaction centers. Thus the binding of the carotenoid to the R 26 reaction centers most likely occurs at the same proteic site as in the wild-type reaction centers. This site shows selectivity towards the nature of carotenoids, and has the same sterical requirement as in the wild type, leading to the observed all-trans to cis isomerisation.