222 Biochimica et BiophysicaActa. 976 (1989) 222-232 Elsevier BBABIO 43060 Monomeric bacteriochlorophyll is required for the triplet energy transfer between the primary donor and the carotenoid in photosynthetic bacterial reaction centers Harry A. Frank and Carol A. Violette Department of Chemistry, UnioersiO, of Connecticut. Storrs, CT (U.S.A.) (Received 11 January 1989) Key words: Borohydride;Carotenoid; Reaction center; Triplet energy transfer; ( Rb. sphaeroides) Reaction centers from the carotenoidless mutant Rb. sphaeroides R26 were treated with sodium borohydride which is known to remove one of the accessory monomeric bacteriochlorophylls (BB). Subsequently, the carotenoid, spheroi- dene, was incorporated into the modified reaction centers, it is demonstrated by optical absorption and circular dichroism experiments that spheroidene, reconstituted into the sodium 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 untreated (native) Rb. sphaeroides R26 reaction centers. Transient optical and electron spin resonance spectroscopic data indicate that unless the accessory B B is present, the primary donor-to-carotenoid triplet energy transfer reaction is inhibited. These observations provide direct evidence for the involvement of the accessory B n in the triplet energy transfer pathway. Introduction The crystallization and X-ray analysis of the caro- tenoid-containing photosynthetic bacterial reaction centers of Rhodopseudomonas oiridis [1-3] and Rhodo- bacter sphaeroides wild-type strain 2.4.1 [4,5] have pro- vided a structural basis from which to understand the controlling features of primary donor-to-carotenoid tri- plet energy transfer. The coordinate refinements carried out on these reaction centers indicate that the carotenoid is located in essentially the same place in both protein complexes. Spheroidene in Rb. sphaeroides and 1,2-di- hydroneurosporene in Rps. viridis were found to lie on the so-called 'inactive' branch (the 'M subunit' or 'B' side, thought to be inactive in electron transfer) of the reaction center, approx. 4 ,~, from the bacteriochloro- phyll (BChl) monomer (BB) associated with that branch, and approx. 10.5 A, away from the primary donor [3-5]. Despite the similarity in the locations of the carotenoids in Rb. sphaeroides and Rps. viridis reaction centers, there are profound differences in their triplet energy Abbreviations: BChl, bacteriochlorophyll; CD, circular dichroism; ESR, electron spin resonance. Correspondence: H.A. Frank, Department or Chemistry, University of Connecticut U-60, 215 Glenbrook Road, Storrs, CT 06269 U.S.A. transfer properties. In Rps. viridis there is no carotenoid triplet formation in the reaction center at any tempera- ture [6,7]. On the other hand, the carotenoid, spheroi- dene, in reaction centers of Rb. sphaeroides wild type strain 2.4.1 accepts triplet energy from the primary donor with very high quantum yield at temperatures above 35 K [8]. Given the fact that the triplet energy transfer reaction is important in photoprotecting the apparatus [9], it is interesting to ask why spheroidene is an efficient triplet trap in Rb. sphaeroides whereas 1,2-dihydroneurosporene in Rps. oiridis is not. Explaining how the triplet energy is transferred and what energy states are involved in the process has been the focus of several experiments carried out on reaction centers [8,10-16], antenna proteins [17-19], and syn- thetic model compounds [17,20-24]. The studies of co- valently linked, synthetic carotenoporphyrin systems have revealed a close distance (essentially van der Waals contact) requirement for high quantum yield triplet energy transfer between spatially fixed porphyrin donor/carotenoid acceptor pairs. Thus, if spheroidene in the reaction center of Rb. sphaeroides 2.4.1 is approx. 10.5 ,~ away from the primary donor, as the X-ray structures indicate, then how is this reconciled with the carotenoporphyrin studies which indicate close pigment proximity is needed for this process? One way in which the data can be reconciled is to invoke the accessory B B 0005-2728/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)