Primary donor-to-carotenoid

C. The Mechanism of Triplet Energy Transfer from the Primary Donor-to-Carotenoids in Chemically... 

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. 

The spectroscopic data presented herein provide direct experimental evidence that the monomeric bacteriochlorophyll is involved in the primary donor-to-carotenoid triplet energy transfer process. 

The reaction center contains one carotenoid molecule, except in the carotenoidless strain R-26 of Rb. sphaeroides. Both the spheroidene in Rb. sphaeroides and the 1,2-dihydroneurosporene in Rp. viridis assume the 15,15 -cA configuration and are located near the Bb molecule (see Figs. 9 and 10). The protein environment around the carotenoid consists of a large number of aromatic residues, which probably impose strong steric constraints on the carotenoid, and may account for the red shift in the absorption spectrum of the carotenoid relative to that of the free carotenoid. The proximity of the carotenoid to Bb suggests that the latter could serve as a conduit for the transfer of triplet-state energy from the primary donor to the carotenoid. 

Kolaczkowski (1989) incorporated perdeutero-spheroidene into protonated and deuterated RCs and explored the role of vibrational terms in the mechanism of triplet enagy transfer from the primary donor to the carotenoid. Mechanisms of triplet energy transfer have been postulated to involve specific electronic and/or vibrational states and/or structural changes in the protein to account for the activated nature of the process (Frank et al., 1983, 1993a, 1996 Takiff and Boxer, 1988a,b Kolaczkowski, 1989 Fous and Hoff, 1989). 

Further work using time-resolved EPR and magnetophotoselection (MPS), using plane-polarized light to excite the triplet state, gave information on the orientation of the optical transition dipole axes relative to the principal axes of the triplet state. By this technique the transition moments of the primary donor"6, the carotenoid in the bRC"7 and the bacteriopheophytin in the inactive B branch 4>0"8 were determined. 

The protein complex of T. elongatus consists of 12 subunits that contain 96 Chi a and 22 carotenoid molecules, 3 [4Fe4S] centres and 2 phylloquinone (vitamin K,) molecules (for molecular structures see Fig. 2). The cofactors of the ET chain are arranged in two branches as pairs of molecules related by a pseudo-C2 axis. After light excitation an electron is donated from the primary donor P700, a pair of chlorophylls, to monomeric chlorophyll a (acceptor A0), phylloquinone (A() and the 3 iron-sulfur centres (F , Oa and B). It has been controversially discussed in the literature whether both highly symmetric pigment branches are... 

Two bacteriochlorophyll monomers, and Bg, are located next to the primary donor D, but they are buried deeper in the membrane. Their positions are fixed by helices B, C, D, and de of the L- and M-subunits, respectively. How these so-called accessory bacteriochlorphylls are involved in the ET has been the subject of a long debate (Holzapfel et al., 1990 Kirmaier and Holten, 1991). Some evidence for their function as true electron carriers has been provided by subpicosecond absorption spectroscopy (Arlt et al., 1993 Zinth et al., 1996). The Bg molecule facilitates the triplet energy transfer between D and the carotenoid (Frank and Violette, 1989). B and Bg follow the local Cj symmetry. Their tetrapyrrole rings are superimposed by a rotation (M on L) of -175.8 (Deisenhofer and Michel, 1989a,b) which is not as perfect as for the D /Dgpair. As in the case of Dg,the phytyl side chain of Bg interacts with the M-subunit... 

Boucher et al. (1977) were the first to report that exogenous carotenoids could be incorporated into RCs from carotenoidless bacteria. Using the G9 strain from Ri. rubrum, these authors demonstrated that four carotenoids, spirilloxanthin, spheroidene, spheroidenone and chloroxanthin could be incorporated with nearly 1 1 mol ratios with respect to the primary donor (P 870). The authors showed that the bound carotenoids protected BChl against photodynamic bleaching. An analysis of the absorption and circular dichroism (CD) spectra of the bound carotenoids lead the authors to conclude that the carotenoids adopted a central mono-c/v configuration, a conclusion later confirmed by X-ray diffraction studies on Rhodopseudomonas viridis and Rb. sphaeroides (Yeates et al., 1988 Amoux, 1989 Deisenhofer and Michel, 1989 Ermler et al. 1994 Chapter 6, Fritsch). 

Work by Farhoosh et al. (1997) sought to explore systematically the effect of changing the extent of r-electron conjugation of the carotenoid bound in the RC and its consequence on the yield and dynamics of triplet energy transfer from the primary donor. The authors analyzed three carotenoids, spheroidene,... 

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15 -cA-spheroidene, were studied by absorption, fluorescence, CD, fast transient absorption and EPR spectroscopies in solution and after incorporation into the RC of Rb. sphaeroides R-26.1. High performance liquid chromatography (HPLC) purification of the synthetic molecule reveal the presence of several Ai-cis geometric isomers in addition to the mono-c/x isomer of locked-15,15 -c/x-spheroidene. In solution, the absorption spectrum of the purified mono-cA sample was red-shifted and showed a large c/x-peak at 351 nm compared to unlocked all-spheroidene. Spectroscopic studies of the purified locked-15,15 -mono-c/x molecule in solution revealed a more stable manifold of excited states compared to the unlocked spheroidene. Molecular modeling and semi-empirical calculations revealed that geometric isomerization and structural factors affect the room temperature spectra. RCs of Rb. sphaeroides R-26.1 in which the locked-15,15 -c/x-spheroidene was incorporated showed no difference in either the spectroscopic properties or photochemistry compared to RCs in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 RCs which naturally contain spheroidene. The data indicate that the natural selection of a c/x-isomer of spheroidene for incorporation into native RCs of Rb. sphaeroides wild type strain 2.4.1 was probably more determined by the structure or assembly of the RC protein than by any special quality of the c/x-isomer of the carotenoid that would affect its ability to accept triplet energy from the primary donor or to carry out photoprotection. 

X-ray diffraction methods have provided the detailed structures of the reaction centers from two carotenoid-containing puiple photosynthetic bacterial species, Rhodopseudomonas viridis [1] and Rhodobacter sphaeroides wild type strain 2.4.1 [2]. The coordinates of these structures indicate that the reaction center-bound carotenoid is located in the M subunit, close ( 4A) to the accessory bacteriochlorophyll monomer on the M subunit side and -lO.SA edge-to-edge distance from the primary donor. These structures suggest an involvement of the M-side monomeric bacteriochlorophyll in triplet-triplet energy transfer, but there has been no direct experimental verification of this hypothesis. 

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