Photosynthesis Research 59: 223–230, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 223 Regular paper Effects of mutagenesis on the detailed structure of spheroidenone in the Rhodobacter sphaeroides reaction centre examined by resonance Raman spectroscopy Andrew Gall 1 , Justin P. Ridge 2 , Bruno Robert 1,∗ , Richard J. Cogdell 3 , Michael R. Jones 2 & Paul K. Fyfe 3,4 1 Section de Biophysique des Prot´ eines et des Membranes, DBCM-CEA and URA 2096 CNRS, Centre d’Etudes de Saclay 91191Gif-sur-Yvette Cedex, France; 2 Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, United Kingdom; 3 Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom; 4 Present address: Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, United Kingdom; ∗ Author for correspondence and/or reprints Received 22 September 1998; accepted in revised form 6 January 1998 Key words: mutagenesis, pigment-protein, spheroidenone, structure, Raman spectroscopy Abstract The effect of mutagenesis on the detailed conformation of the carotenoid cofactor of the bacterial reaction centre has been examined using resonance Raman spectroscopy. Four single site mutations were made, removing polar residues that line the binding pocket for spheroidenone in the reaction centre from Rhodobacter sphaeroides. All of the mutations caused changes in the relative intensity of bands in the ν 2 frequency region of the carotenoid res- onance Raman spectrum, suggesting a change in the geometry of the central 15,15 ′ -cis bond of the spheroidenone. In addition, increased splitting of the ν 1 vibrational modes in two of the mutant RCs indicated a reduction of the effective conjugation length of the spheroidenone, possibly due to an increased distortion from a planar geometry along the C=C backbone of the spheroidenone. These changes in the detailed conformation of the reaction centre carotenoid do not affect the optical properties of the cofactor, and are beyond the limits of detection of X-ray crystallography as currently applied to the bacterial reaction centre. Abbreviations: Rb.– Rhodobacter; RC – reaction centre; WT – wild-type Introduction The principal function of the carotenoid cofactor of the Rhodobacter (Rb.) sphaeroides reaction centre (RC) is to protect the protein from photo-oxidative damage (Cogdell and Frank 1987; Frank and Cogdell 1993). The carotenoid achieves this by quenching the energy of bacteriochlorophyll triplet states before they can re- act to form singlet oxygen. It has been known for many years that the conformation of the carotenoid bound within the RC is different from the all-trans conform- ation found in solution (Boucher et al. 1977; Lutz et al. 1976). On the basis of resonance Raman spectro- scopy, it has been concluded that the spheroidene that is bound to the Rb. sphaeroides RC when the organism is grown under anaerobic conditions adopts a 15,15 ′ - cis conformation (Koyama et al. 1982). When Rb. sphaeroides is grown under semiaerobic conditions spheroidene is replaced by spheroidenone, which also adopts a 15,15 ′ -cis conformation when bound to the RC (Lutz et al. 1978). X-ray crystallography of the Rb. sphaeroides RC has shown that both spheroidene and