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.     

Analysis by exciton theory of the optical properties of the Chloroflexus aurantiacus reaction center

The optical properties of the reaction center of the filamentous green bacterium Chloroflexus aurantiacus, that contains three bacteriochlorophyll (BChl) a and three bacteriopheophytin (BPh) a molecules, were analyzed in the near-infrared region with the aid of exciton theory. The coordinates obtained from the X-ray analysis of the reaction center of Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) were used for the geometry of the reaction center of C. aurantiacus, with the replacement of one of the ‘accessory’ BChl molecules by BPh. The results were found to be in good agreement with experimental low-temperature absorption spectra, linear and circular dichroism and fluorescence polarization spectra and lead to the following conclusions. The allowed, low-energy exciton transition of the primary electron donor (P-865) is located at 887 nm and carries the dipole strength of approx. two BChl a monomers; the high-energy exciton transition, around 790 nm, is mixed with wave functions of other pigments, which explains its relatively small angle with respect to the 887 nm transition. The optical transition of the accessory BChl a molecule near 812 nm has some contribution of the BChls that constitute P-865. This can account for the experimentally observed reorientation and shift of this transition upon oxidation of P-865. Two of the BPh molecules are located on the same (probably the M) polypeptide subunit and show a clear splitting of absorption bands (11 nm) due to exciton coupling; the single BPh on the opposite branch shows hardly any exciton shift. Similar calculations for reaction centers of purple bacteria that contain four BChl a and two BPh a molecules resulted in a very low dipole strength for the high-energy transition of the primary donor due to antisymmetric mixing with both accessory BChl a wave functions and gave very little splitting of the absorption bands of BPh a. Our results indicate that the arrangement of the chromophores in reaction centers of C. aurantiacus is very similar to that in purple bacteria. The functional L-chains of the reaction centers of purple and filamentous green bacteria consist of pigments of the same type in a probably very similar arrangement.     

Antenna and reaction-center processes upon picosecond-flash excitation of membranes of the green photosynthetic bacterium Chloroflexus aurantiacus

The formation and decay of antenna-excited states and the primary charge separation in membranes of the green photosynthetic bacterium Chloroflexus aurantiacus were studied by means of picosecond absorbance difference spectroscopy. After chemical oxidation of the primary electron donor, a 35 ps excitation pulse at 532 nm produced singlet- and triplet-excited states of carotenoid and of bacteriochlorophyll a. Excitation of bacteriochlorophyll a caused a bleaching of its Q_y absorption band and induced a blue shift of several neighboring bacteriochlorophyll molecules. The singlet-excited state decayed biphasically with lifetimes of about 200 ps and 1.2 ns. A decrease in the lifetime at increasing flash intensity was attributed to singlet-singlet annihilation. In the presence of active reaction centers also the primary-charge separation and secondary electron transfer were observed. The charge separation consisted of the transfer of an electron from the primary donor, P-865, to the primary-acceptor complex of bacteriopheophytin a and bacteriochlorophyll a. Electron transfer to a secondary acceptor occurred with a time constant of 400 ± 50 ps, which is about 30% longer than had been observed with isolated reaction centers (Kirmaier, C., Holten, D., Mancino, L.J. and Blankenship, R.E. (1984) Biochim. Biophys. Acta 765, 138–146). When this secondary acceptor was prereduced chemically, the lifetime of the primary radical pair increased to 10 ns or more.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Study of the long-wavelength fluorescence band at 920 nm of isolated reaction centers of the photosynthetic bacterium Rhodopseudomonas spaeroides R-26 with fluorescence-detected magnetic resonance in zero field

The triplet state of isolated reaction centers of Rhodopseudomonas sphaeroides R-26 has been studied by fluorescence-detected electron spin resonance in zero magnetic field (FDMR) at 4.2 K. The sign of the FDMR resonance monitored at the long-wavelength fluorescence band is positive (fluorescence increase); this confirms the earlier interpretation (Hoff, A.J. and Gorter de Vries, H. (1978) Biochim. Biophys. Acta 503, 94–106) that the negative sign of the FDMR resonance of the reaction center triplet state in whole bacterial cells is caused by resonant transfer of the singlet excitations from the antenna pigments to the trap. By monitoring the FDMR response as a function of the wavelength of fluorescence, we have recorded microwave-induced fluorescence spectra. In addition to the positive microwave-induced fluorescence band peaking at 935 nm, at 905 nm a negative band was found. The resonant microwave frequencies for these two bands, i.e., the values of the zero-field splitting parameters |D| and |E| of the triplet state being monitored, were different, those of the 905 nm microwave-induced fluorescence band being identical to the resonant microwave frequencies measured with absorption-detected zero-field resonance (Den Blanken, H.J., Van der Zwet, G.P. and Hoff, A.J. (1982) Chem. Phys. Lett. 85, 335–338), a technique that monitors the bulk properties of the sample. From this result and its negative sign, we tentatively attribute the 905 nm microwave-induced fluorescence band to a small (possibly less than 1%) fraction of antenna bacteriochlorophylls that are in close contact with the trap. The positive 935 nm microwave-induced fluorescence band with resonant microwave frequencies deviating from the bulk material is ascribed to a minority of primary donor bacteriochlorophyll dimers, which have a higher than normal fluorescence yield because of a somewhat slower charge-separation reaction. Is it likely that practically all long-wavelength fluorescence of isolated reaction centers stems from such impaired reaction centers.     

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy

Reaction centers from two species of purple bacteria, Rhodospirillum rubrum and Rhodospirillum centenum, have been characterized and compared to reaction centers from Rhodobacter sphaeroides and Rhodobacter capsulatus. The reaction centers purified from these four species can be divided into two classes according to the spectral characteristics of the primary donor. Reaction centers from one class have a donor optical band at a longer wavelength, 865 nm compared to 850 nm, and an optical absorption band associated with the oxidized donor at 1250 nm that has a larger oscillator strength than reaction centers from the second class. Under normal buffering conditions, reaction centers isolated from Rb. sphaeroides and Rs. rubrum exhibit characteristics of the first class while those from Rb. capsulatus and Rs. centenum exhibit characteristics of the second class. However, the reaction centers can be converted between the two groups by the addition of charged detergents. Thus, the observed spectral differences are not due to intrinsic differences between reaction centers but represent changes in the electronic structure of the donor due to interactions with the detergents as has been confirmed by recent ENDOR measurements (Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Biochemistry 33: 12077–12084). The oxidation midpoint potential for the donor has values of 445 mV, 475 mV, 480 mV and 495 mV for Rs. rubrum, Rs. centenum, Rb. capsulatus, and Rb. sphaeroides, respectively. Despite this range of values for the midpoint potential, the decay rates of the stimulated emission are all fast with values of 4.1 ps, 4.5 ps. 5.5 ps and 6.1 ps for quinone-reduced RCs from Rs. rubrum, Rb. capsulatus, Rs. centenum, and Rb. sphaeroides, respectively. The general spectral features of the initial charge separated state are essentially the same for the four species, except for differences in the wavelengths of the absorption changes due to the different donor band positions. The pH dependence of the charge recombination rates from the primary and secondary quinones differ for reaction centers from the four species indicating different interactions between the quinones and ionizable residues. A different mechanism for charge recombination from the secondary quinone, that probably is direct recombination, is proposed for RCs from Rs. centenum.Abbreviations RC reaction center - P bacteriochlorophyll dimer - H bacteriopheophytin - Q quinone - Rb Rhodobacter - Rs Rhodospirillum - Rps Rhodopseudomonas - EDTA (ethylenediamine)tetraaceticacid - LDAO N,N-dimethyl-dodecylamine-N-oxide - CTAB cetyltrimethylammonium bromide - DOC deoxycholate - Tris Tris-(hydroxymethyl)aminomethane - ns nanosecond - ps picosecond - fs femtosecond     

The absence of a spectroscopically resolved intermediate state P+B− in bacterial photosynthesis

Reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been excited either in the bacteriopheophytin band at 760 nm or in the accessory bacteriochlorophyll (B) band around 800 nm with laser pulses of 150 fs duration. Upon monitoring in the absorption band of the primary donor (P) at 860 nm, ultrafast energy transfer is observed which leads to the excited state of P in less than 100 fs. A transient bleaching recovering in 400 ± 100 fs is specifically detected upon excitation and observation in the 800 nm absorption band of B. However, upon direct excitation of P in the near infrared and using either normal or borohydride-treated reaction centers, we have found no spectral or kinetic evidence indicating the presence of a transient intermediate state such as P⁺B⁻.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.     

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.     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.     

An alternative pathway of light-induced transmembrane electron transfer in photosynthetic reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P₈₇₀. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical B_A ^– at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor Q_A, with the primary electron donor P₈₇₀ remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PB_AH_AQ_A ^–.     

Temperature and detection-wavelength dependence of the picosecond electron-transfer kinetics measured in Rhodopseudomonas sphaeroides reaction centers. Resolution of new spectral and kinetic components in the primary charge-separation process

We have examined the temperature dependence of the rate of electron transfer to ubiquinone from the bacteriopheophytin (BPh) that serves as an initial electron acceptor (I) in reaction centers of Rhodopseudomonas sphaeroides. The kinetics were measured from the decay of the 665-nm absorption band of the reduced BPh (BPh⁻ or I⁻) and from the recovery of the BPh band at 545 nm, following excitation of reaction centers in polyvinyl alcohol films with 30-ps flashes. The measured time constant decreases from 229 ± 25 ps at 295 K to 97 ± 8 ps near 100 K and then remains constant down to 5 K. The temperature dependence of the kinetics can be rationalized on the assumption that the reaction results in changes in the frequencies of numerous low-energy nuclear (vibrational) modes of the electron carriers and/or the protein. The kinetics measured in the absorption bands near 765 and 795 nm show essentially the same temperature dependence as those measured at 545 or 665 nm, but the time constants vary with detection wavelength. The time constant measured in the 795-nm region (70 ± 10 ps at 5 and 76 K) is shorter than that seen in the absorption bands of the BPh; the time constant measured at 758 nm is longer. Time constants measured with reaction centers in solution at 288 K also vary with the detection wavelength. These results can be explained on the assumption that the absorption changes measured at some wavelengths reflect nuclear relaxations rather than electron transfer. The absorption changes at 795 nm probably reflect a relaxation of the bacteriochlorophyll molecules that are near neighbors of the BPh and the primary electron donor (P). Those near 530 and 755 nm probably are due to the second BPh molecule, which does not appear to undergo oxidation or reduction.     

Photoreactions of bacteriopheophytins and bacteriochlorophylls in reaction centers of Rhodopseudomonas sphaeroides and Chloroflexus aurantiacus

A comparison of spectral properties of reaction centers from Chloroflexus aurantiacus and Rhodopseudomonas sphaeroides (R-26) is reported. Treatment of reaction centers from Rps. sphaeroides with NaBH₄ leads to a decrease of the dipole strength of the 800-nm band by factor of approx. 1.75-1.95 and to the formation of new bacteriopheophytin, BPh-715, which is almost completely removed during the purification of reaction centers. The modification of the reaction centers does not change the quantum yield of P photooxidation and the spectrum of BPh-545 (H₁) photoreduction which includes the changing of the 800-nm band. This implies the preservation of the photoactive chain P-B₁-H₁-Q_A (where B₁ is the bacteriochlorophyll (BChl)-800 molecule situated between P and H₁) and the modification of the second BChl-800 (B₂). The preparation of modified reaction centers is a mixture of at least three types of reaction centers with different contents of B₂ and of the second BPh (H₂). Some of the reaction centers (5-25%) contain the original B₂ and H₂ molecules (type I). In the CD spectrum of modified reaction centers a decrease of the 800-nm band and the appearance of a positive band at 765 nm is observed. This spectrum is similar to the CD spectrum of Chloroflexus reaction centers containing 3 BPh's and 3 BChl's. This implies that in some (approx. 40%) of the modified Rps. sphaeroides reaction centers (type II) B₂ has been replaced by BPh a which interacts with H₂. Probably some of the modified reaction centers (approx. 40%) have lost both B₂ and H₂ (type III). The modification of reaction centers leads to a considerable decrease of the CD bands at 800 (+) nm and 810 (−) nm and to a decrease of the absorbance changes near 800 nm in the difference absorption spectrum of the oxidation of P. The data are interpreted in terms of the interaction between P and B₁ molecules which gives two transitions at 790-800 and 810 nm with different orientations in modified Rps. sphaeroides as well as in Chloroflexus reaction centers. Similar transitions are observed for the interaction between P and B₂. The spectral analysis shows the existence of two chains P-B₁-H₁, and P-B₂-H₂ in which the distances between the centers of molecules are 1.3 nm or less.     

The carotenoid band shift in reaction centers 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.     

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.     

Linear-dichroic triplet-minus-singlet absorbance difference spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum,Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1

For the first time, linear-dichroic triplet-minus-singlet (LD-(T - S)) spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum, Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1 have been measured using an extension of the technique of absorbance-detected magnetic resonance (ADMR) of the triplet state. For all bacteria studied the LD-(T - S) spectra exhibit a bleaching of the long-wavelength absorbance band that is either split or has a clear shoulder to longer wavelengths. The components are approximately parallel-polarized, indicating that they do not form an exciton pair. Around 800 nm a band appears with a width of about 7 nm, which does not form part of a band shift and that may be attributed to an appearing monomer band. Small features in the LD-(T - S) spectra at both sides of this band are well explained by band shifts of the two components of the 800 nm reaction center absorption band. The transition moment of the component at about 818 nm in reaction centers of Rps. sphaeroides R-26 is at an angle larger than 55° with both the x and the y triplet spin axes. In none of the bacteria do we find evidence for the bleaching of an exciton component of P-860 near 810 nm.     

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm (Rps. sphaeroides) and 810–890 nm (Rps. viridis). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.     

Structure of Chloroflexus aurantiacus reaction center: Photoselection at low temperature

Photoselection experiments have been performed on isolated Chloroflexus aurantiacus reaction centers at 20 K. Our data show that the average angle between the ground state BPh Q_y transitions and the 890 nm transition is approx. 50°. Only two BPh Q_y transitions are affected by the charge separation. These two transitions are perpendicular to the long-wavelength band of the primary donor. The ground state of the 813 nm transition makes an angle of 35° with the dimer absorption band. The polarization ratio of the light-induced absorption decrease at 815 nm is not consistent with that decrease being due solely to an electrochromic bandshift of the 813 nm transition.     

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.-C. Lambry and J.-L. Martin (1994) Biochemistry 33, 6750-6757] and in Qy absorption band of the primary electron acceptor, bacteriochlorophyll monomer B(A) [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293-7298] with a set of fundamental frequencies in the range of 10-300 cm(-1). We have found that in pheophytin-modified RCs, the fs oscillations with frequency around 130 cm(-1) observed in the P*-stimulated emission as well as in the B(A) absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer B(A)-. These results are discussed in terms of a reversible electron transfer between P* and B(A) induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+B(A)-, when a maximal value of the Franck-Condon factor is created.