Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g_F, where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b_P, where P is phytyl, rather than the native BChl a_P, we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g_P. Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG^Rs gene from Rba. sphaeroides was replaced with bchG^Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g_F. The construction of a strain producing BChl g_F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g_F.     

Formation and decay of radical-pair state P+I− in Chloroflexus aurantiacus reaction centers

We have examined the room temperature kinetics of the absorption changes associated with the formation of state P⁺I⁻ (P⁺BPh⁻) and its subsequent decay to state P⁺Q⁻_A in reaction centers from Chloroflexus aurantiacus. Our data, acquired using 30-ps excitation flashes, strongly suggest that formation of P⁺I⁻ (P⁺BPh⁻) takes longer in Chloroflexus than in reaction centers from Rhodopseudomonas sphaeroides. The reduction of the photoactive bacteriopheophytin (BPh) could take as long as 13 ps. Absorption changes different from those due to P⁺I⁻ are observed early in the excitation flash, but the detailed identity of the transient remains unclear. We also find that the kinetics observed subsequent to P⁺I⁻ formation differ with detection wavelength. The time constant measured in the anion band (I⁻) at 655 nm is 324 ± 20 ps and probably reflects the rate of electron transfer from I⁻ (BPh⁻) to Q_A. However, the kinetics measured in the BPh ground-state absorption bands are slightly longer: 365 ± 19 and 367 ± 21 ps at 538 and 760 nm, respectively. At 810 nm, a wavelength normally associated with the monomeric bacteriochlorophyll (BChl) in the Chloroflexus reaction center, a slightly faster (281 ± 19 ps) time constant is observed. This detection-wavelength dependence of the kinetics is similar to that observed recently in Rps. sphaeroides reaction centers. Comparison of these results suggests that the kinetics observed in the ground-state absorption bands of the BPhs and BChls in Chloroflexus may contain contributions from readjustments of the pigments and/or protein in response to the charge separation process.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

Light-induced red shift of the Qx absorption band of light-harvesting bacteriochlorophyll in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the Q_x band(s) of the light-harvesting bacteriochlorophyll (BChl) (λ_max ~590 nm) shifts to the red in response to a light-induced membrane potential, as indicated by the characteristics of the light-minus-dark difference spectrum. In green strains, containing light-harvesting complexes I and II, and one or more of neurosporene, methoxyneurosporene, and hydroxyneurosporene as carotenoids, the absorption changes due to the BChl and carotenoid responses to membrane potential in the spectral region 540–610 nm are comparable in magnitude and overlap with cytochrome and reaction center absorption changes in coupled chromatophores. In strains lacking carotenoid and light-harvesting complex II, the BChl shift absorption change is relatively smaller, due in part to the lower BChl/reaction center ratio.In the carotenoid-containing strains, the peak-to-trough absorption change in the BChl difference spectrum is 5–8% of the peak-to-trough change due to the shift of the longest-wavelength carotenoid band, although the absorption of the BChl band is 25–40% of that of the carotenoid band. The responding BChl band(s) does not appear to be significantly red-shifted in the dark in comparison to the total BChl Q_x band absorption.     

Orientation of the chromophores in the reaction center of Rhodopseudomonas viridis. Comparison of low-temperature linear dichroism spectra with a model derived from X-ray crystallography

Linear dichroism (LD) and absorption (A) spectra of reaction centers from Rhodopseudomonas viridis included in the native chromatophores or reconstituted in planar aggregates have been recorded at 10 K. The samples were oriented in squeezed polyacrylamide gels and the primary donor P was in the reduced or (chemically) oxidized state. The LD spectra of reaction centers in these two states are in favor of a dimeric model of P in which excitonic coupling between the two non-parallel Q_Y transitions leads to a main transition at 990 nm (parallel to the membrane plane) and another one of smaller oscillator strength at 850 nm (tilted at approx. 60° out of the membrane plane). These assignments are in close agreement with the ones proposed in a previous LD study at room temperature (Paillotin, G., Verméglio, A. and Breton, J. (1979) Biochim. Biophys. Acta 545, 249–264). The main Q_X excitonic component of P has a broad absorption peaking at 620 nm and it corresponds to dipoles exhibiting the same orientation as those responsible for the 850 nm transition. On the basis of the present LD study and of CD data of chemically oxidized-minus-reduced reaction centers, we proposed that the minor Q_X excitonic component of P is oriented close to the membrane plane and absorbs around 660 nm. The two monomeric bacteriochlorophylls exhibit a positive LD for both their Q_Y transitions (unresolved at 834 nm) and their Q_X transitions (resolved at 600 and 607 nm), indicating that the planes of these molecules are only slightly tilted out of the membrane plane. The two bacteriopheophytins exhibit strong negative LD with identical LD/A values for their Q_Y transitions (resolved at 790 and 805 nm) and small positive LD for their Q_X transitions (resolved at 534 and 544 nm), demonstrating that these two molecules are strongly tilted out of the membrane plane with each of the Q_Y transitions tilted at approx. 50° out of that plane. A comparison of these LD data with the structural model derived from X-ray crystallography (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) clearly suggests that a good agreement exists between the results of the two techniques under the following conditions: (i) the C-2 symmetry axis of the reaction center runs along the membrane normal; (ii) excitonic coupling is present only in the primary donor special pair; and (iii) the direction of the optical transitions of the monomeric bacteriochlorophylls and of the bacteriopheophytins is not significantly perturbed by the interactions among the pigments. In addition, a carotenoid is detected in the isolated reaction center with an orientation rather perpendicular to the C-2 symmetry axis. Finally, a comparison of these data with similar ones obtained on the bacteriochlorophyll a-containing reaction center of Rhodopseudomonas sphaeroides 241 points towards a geometrical arrangement of the chromophores which is indistinguishable from the one observed in the reaction center of Rps. viridis.     

Pressure effects on the photochemistry of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis

Electron-transfer reactions and triplet decay rates have been studied at pressures up to 300 MPa. In reaction centers from Rhodobacter sphaeroides R-26, high pressure hastened the electron transfers from both the primary and secondary quinones (Q_A and Q_B) to the primary electron donor bacteriochlorophyll, P. Motion of Q_A between two sites, one nearer to P and the other nearer to Q_B, could account for these pressure effects. In reaction centers from Rhodopseudomonas viridis, charge recombination was slowed by high pressure. Decay rates were also studied for the triplet state, P^R. In Rb. sphaeroides R-26 with Q_A reduced with Na₂S₂O₄, the decay was hastened by pressure. This could be explained if P^R decays through a charge-transfer triplet state, or if the decay kinetics of P^R are sensitive to the distance between P and Q_A⁻. In Rps. viridis reaction centers, and in Rb. sphaeroides reaction centers that were depleted of Q_A, the lifetime of P^R was not altered by pressure.     

The reaction center profile structure derived from neutron diffraction

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 Å resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 Å resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 Å.     

Quinone transport in the closed light-harvesting 1 reaction center complex from the thermophilic purple bacterium Thermochromatium tepidum

Redox-active quinones play essential roles in efficient light energy conversion in type-II reaction centers of purple phototrophic bacteria. In the light-harvesting 1 reaction center (LH1-RC) complex of purple bacteria, Q_B is converted to Q_BH₂ upon light-induced reduction and Q_BH₂ is transported to the quinone pool in the membrane through the LH1 ring. In the purple bacterium Rhodobacter sphaeroides, the C-shaped LH1 ring contains a gap for quinone transport. In contrast, the thermophilic purple bacterium Thermochromatium (Tch.) tepidum has a closed O-shaped LH1 ring that lacks a gap, and hence the mechanism of photosynthetic quinone transport is unclear. Here we detected light-induced Fourier transform infrared (FTIR) signals responsible for changes of Q_B and its binding site that accompany photosynthetic quinone reduction in Tch. tepidum and characterized Q_B and Q_BH₂ marker bands based on their ¹⁵N- and ¹³C-isotopic shifts. Quinone exchanges were monitored using reconstituted photosynthetic membranes comprised of solubilized photosynthetic proteins, membrane lipids, and exogenous ubiquinone (UQ) molecules. In combination with ¹³C-labeling of the LH1-RC and replacement of native UQ₈ by ubiquinones of different tail lengths, we demonstrated that quinone exchanges occur efficiently within the hydrophobic environment of the lipid membrane and depend on the side chain length of UQ. These results strongly indicate that unlike the process in Rba. sphaeroides, quinone transport in Tch. tepidum occurs through the size-restricted hydrophobic channels in the closed LH1 ring and are consistent with structural studies that have revealed narrow hydrophobic channels in the Tch. tepidum LH1 transmembrane region.     

The location of redox centers in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex by resonance X-ray diffraction

The technique of resonance X-ray diffraction (Blasie, J.K. and Stamatoff, J. (1981) Annu. Rev. Biophys. Bioeng. 10, 451–452) utilizing synchrotron radiation was used to determine the locations of the cytochrome c heme iron atom and the photosynthetic reaction center iron atom within the profile of a reconstituted membrane. The accuracy of these determinations was better than ±2 ?. The cytochrome c heme iron atom → reaction center iron atom vector was determined to have a magnitude of approx. 44 ? projected onto the membrane profile and to span most of the lipid hydrocarbon core of the membrane profile. Since the reaction center iron atom interacts magnetically with the primary quinone electron acceptor Q_I over a distance of less than 10 ?, the primary light-induced electron-transfer reactions for this system generate the electric charge separation between oxidized cytochrome c⁺ and Fe-Q^?_I across most (approx. $\text{2}\text{3}$) of the membrane profile including most or all of the lipid hydrocarbon core of the membrane.     

Free-energy change accompanying the reduction of the reaction center secondary quinone in Rhodopseudomonas sphaeroides chromatophores

The standard free-energy change accompanying the electron transfer from Q_A to Q_B was estimated from the intensity of the delayed fluorescence in chromatophores of Rhodopseudomonas sphaeroides. The value of 120 meV (at pH 8) suggests that Q_B⁻ is more stable in the chromatophore membrane than in the isolated reaction center.     

Differential extraction and structural specificity of specialized ubiquinone molecules in secondary electron transfer in chromatophores from Rhodopseudomonas sphaeroides, Ga

In chromatophores from the facultative photosynthetic bacterium, Rhodopseudomonas sphaeroides, Ga, the function of ubiquinone-10 (UQ-10) at two specialized binding sites (Q_B and Q_Z) has been determined by kinetic criteria. These were the rate of rereduction of flash-oxidized [BChl]₂⁺ through the back reaction, or the binary pattern of cytochrome b₅₆₁ (for the Q_b site), and the rapid rate of rereduction of flash-oxidized cytochrome c, or the relative amplitude of the antimycin-sensitive Phase III ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{~ 1.5}\text{ms}$) of the carotenoid spectral shift induced by a single turnover flash at E_h ~ 100 mV (for the Q_Z site). The phenomenon associated with the two binding sites behaved differently on extraction of UQ from lyophilized chromatophores using isooctane. By this selective extraction procedure it has been possible to show that UQ-10 molecules are required at different concentrations in the membrane for specific redox events in secondary electron transfer. The reduction of cytochrome b occurs in particles which no longer show the phenomena associated with Q_Z, but still possess a large proportion of Q_b, while rapid rereduction of flash-oxidized cytochrome c requires an additional complement of UQ-10 (Q_Z). Extracted particles lacking Q_Z and a large amount of Q_B have been reconstituted with different UQ homologs (UQ-1, UQ-3, and UQ-10). Specific redox events have been studied in reconstituted particles. All UQ homologs act as secondary acceptors from the reaction center; UQ-3 and UQ-10, but not UQ-1, are also able to reconstitute the function of Q_Z as electron donor to cytochrome c. Only UQ-10, however, is able to restore normal rates of the overall cyclic electron transfer induced by a train of flashes, and maximal rates of the light-induced ATP synthesis. The results are interpreted in terms of Q-cycle mechanisms in which quinone and quinol at both the Q_Z and Q_b sites are in rapid equilibrium with the quinone pool.     

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)₂, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)₂ in the first singlet excited state, (BChl)1₂, through the bacteriopheophytin, BPh, to the primary ubiquinone, Q_A. Possible contributions to the field-induced loss of quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation are: (1) a decrease in the free-energy gap between the states (BChl)1₂ and (BChl)^⨥₂BPh^⨪Q_A, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)^⨥₂BPh^⨪Q_A directly or via the (BChl)₂ triplet state to the ground state and (3) an impeded electron transfer from (BChl)^⨥₂BPh^⨪Q_A to (BChl)^⨥₂BPhQ^⨪_A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

Antenna organization and energy transfer in membranes of Heliobacterium chlorum

Absorption, fluorescence emission and fluorescence excitation spectra of membranes of the recently discovered photosynthetic bacterium Heliobacterium chlorum (Gest, H. and Favinger, J.L. (1983) Arch. Microbiol. 136, 11–16) showed that at 4 K at least three spectroscopically different forms of bacteriochlorophyll g (BChl g 778, BChl g 793 and BChl g 808) can be discerned in the antenna system. Efficient energy transfer occurs from the short-wave-absorbing bacteriochlorophylls to BChl g 808. Energy transfer to bacteriochlorophyll, albeit with lower efficiency (70%), also occurred from the main carotenoid, neurosporene, and from a pigment absorbing at 670 nm. The complex structure of the antenna system is also reflected by fluorescence polarization and linear and circular dichroism spectra. Significant circular dichroism was only observed for BChl g 793, and different orientations were observed for the various Q_y transition dipoles, the one of BChl g 808 making a smaller angle with the plane of the membrane than those of the other bacteriochlorophylls.     

Picosecond kinetics in reaction centers of Rps. sphaeroides and the effects of ubiquinone extraction and reconstitution.

Following picosecond light activation, the bacteriochlorophyll and bacteriopheophytin complement of $\text{Rps.}\text{sphaeroides}$ reaction centers depleted of ubiquinone behaves as though it has no primary electron acceptor; the excited intermediary $\text{BChl}\text{BPh}{}^2$ state formed in <10 ps lasts >1 ns. Addition of ubiquinone-10 reconstitutes the very rapid electron transfer rates from the excited intermediary $\text{BCh}\text{BPh}$ state to ubiquinone; the kinetics and rate are similar to that encountered in the untreated reaction centers. Interpretation of the data presented suggests that ubiquinone is the immediate electron acceptor from BPh^?. This is consistent with the model for the primary reactions leading to [(BChl)₂^?BPh]Q^?.     

Electric field effect on absorption spectra of reaction centers of Rb. sphaeroides and Rps. viridis

Comparison of the Stark effect on the Q_y transitions of the pigments in reaction centers of Rb. sphaeroides and Rps. viridis at 77 K shows great similarity in the long-wavelength absorption of the bacteriochlorophyll dimer but differs significantly in the absorption region of the accessory bacteriochlorophylls and bacteriopheophytins.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.