Spheroidene Bound to the Reaction Center

The T state was generated by excitation of the bacteriochlorophyll Qx absorption at 600 nm and subsequent triplet-energy transfer to the carotenoid, and the T Raman spectra were recorded by the use of 532 nm pulses [18]. 

sphaeroides R26 (carotenoidless mutant). The reaction center-bound D0 spheroidene exhibits unique spectral features in both the S0 and Ta states In the S0 state, it exhibits a key Raman line of the 15-ds configuration (at 1239 cm-1), which is a coupled mode of the C15-H and C15 -H in-plane bendings. The Raman profiles of other normal modes are similar to the case of all-trans-spheroi- 

Conformational Changes and the Inversion of Spin-Polarization Identified by Low-Temperature Electron Paramagnetic Resonance Spectroscopy of the Reaction Center-Bound 15-cis-Spheroidene A Hypothetical Mechanism of Triplet-Energy Dissipation [19] 

40 I 3 Mechanisms of Cis-Trans Isomerization around the Carbon-Carbon Double Bonds via the Triplet State 

Magnetic field /103 G Magnetic field /103 G Magnetic field /103 G 

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Fig. 3.16 Changes in the stretching force constants upon excitation from S0 state (open circles) to the T, state (filled circles), showing the triplet-excited region for all-Jrans-spheroidene in solution (a) and for 15-c/s-spheroidene bound to the reaction center (b). In both peripheral parts, some force constants (double circles) were assumed and could not be determined because of the limited number of deuterio species and the lack of observed Raman lines [18]. Circles with a horizontal line indicate that those force constants are the same in both the S0 and T, states.

Fig. 3.17 The S0 and T Raman spectra of undeuterated 15-c/s-spheroidene bound to the reaction center from Rba. sphaeroides 2.4.1 (wild type) and various deuterio derivatives incorporated into the reaction center from Rba. sphaeroides R26 (carotenoidless mutant).

CD spectra of borohydride-treated and native Rb, sphaeroides R26 reaction centers are presented in Fig. 4. For comparison, the CD spectra of Rb, sphaeroides wild type strain 2.4.1 reaction centers are presented in Fig. 5. Because CD is a sensitive probe of the structure and environment of bound chromophores, it provides an opportunity to examine whether or not spheroidene is bound in the same manner for all the complexes. Furthermore, it is known that carotenoids are not optically active unless physically bound to the reaction center protein [8]. This is shown in Fig. 5 which demonstrates that spheroidene, incorporated into Triton X-100 micelles at a concentration equal to that of spheroidene bound to the reaction centers, does not display a CD spectrum. Upon binding to the reaction center protein, spheroidene becomes optically active, presumably either through exciton interactions with the amino acid residues in proximity to the chromophore or by an asymmetry in the carotenoid configuration or conformation, and displays a pronoimced CD spectrum. 

The Triplet-Excited Region of All-trans-Spheroidene in Solution and the Triplet-State Structure of 15-cis-Spheroidene Bound to the Bacterial Reaction Center Determined by Raman Spectroscopy and Normal Coordinate Analysis [18]... 

The triplet-excited region has been spectroscopically identified in terms of carbon-carbon stretching force constants in all-trans-spheroidene in solution and in 15-ris-spheroidene bound to the bacterial reaction center (Fig. 3.16). Large changes in bond order are actually seen in the central part of the conjugated chain in both cases. The conformation of the reaction center-bound 15-cis-spheroidene in the T, state was determined to be (+45°, -30°, +30°) around the cis C15=C15, trans 03=04 and 01=02 bonds. 

The polyene chain of the carotenoid predominantly interacts with aromatic and hydro-phobic amino acids, like it is seen for spheroidene in the reaction center of Rhodobacter sphaeroides (Ermles et al., 1994). Similarly, in the major LHCll of higher plants, the superhelix formed by the transmembrane ahelices A and B seems to be stabilized by two carotenoid molecules bound into the grooves of the superhelix (Kiihlbrandt et al, 1994). Hydrophobic interactions with these carotenoids may be an essential factor for stablizing the arrangement of a helices in this complex, as there are relatively few ionic helix-helix interactions in the major LHCll as compared to other complexes like bacterio-rhodopsin or the bacterial reaction center. 

Here we present experimental evidence that the monomeric bacteriochlorophyll is required for triplet energy transfer from the primary donor to the carotenoid in photosynthetic bacterial reaction centers. Our approach is to use sodium borohydride to extract the monomeric bacteriochlorophyll from the reaction centers of the carotenoidless mutant Rb. sphaeroides R26 [3, 4]. The borohydride treated reaction centers are then reconstituted with the carotenoid, spheroidene [5], and the ability of the reaction center complex to carry out the primary donor-to-carotenoid triplet transfer reaction was examined by transient optical spectroscopy. Steady state optical absorption and circular dichroism (CD) measurements demonstrate diat spheroidene reconstituted into borohydride-treated Rb, sphaeroides R26 reaction centers is bound in a single site, in the same environment and with the same structure as spheroidene reconstituted into native Rb. sphaeroides R26 reaction centers. It is shown herein that the primary donor-to-carotenoid triplet transfer reaction is inhibited in the absence of the accessory bacteriochlorophyll. 

Lutz M, Kleo J and Reiss-Husson F (1976) Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas spheroides. Biochem Biophys Res Commun 69 711-717 Lutz M, Agalidis I, Hervo G, Cogdell RJ and Reiss-Husson F (1978) On the state ofcarotenoids bound to reaction centers of photosynthetic bacteria A resonance Raman study. Biochim Biophys Acta 503 287-303... 

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

Soret region is dominated by the strong contributions of these pigments, as expected from the very low residual cyt c content (< 0.1 heme per reaction center). The only difference concerns the bound carotenoid, which visible absorption bands are blue-shifted by about 7 nm as compared to 15-15 cis-spheroidene in Rb. sphaeroides. In addition, oxidized reaction centers (in presence of ferricyanide) displayed a weak absorption band centered at 1245nm (not shown) attributed as in Rb. sphaeroides to a P+ transition. 

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