Effects of mutagenesis on the detailed structure of spheroidenone in the Rhodobacter sphaeroides reaction centre examined by resonance Raman spectroscopy

The effect of mutagenesis on the detailed conformation of the carotenoid co factor of the bacterial reaction centre has been examined using resonance R aman spectroscopy. Four single site mutations were made, removing polar res idues that line the binding pocket for spheroidenone in the reaction centre from Rhodobacter sphaeroides. All of the mutations caused changes in the r elative intensity of bands in the nu(2) frequency region of the carotenoid resonance Raman spectrum, suggesting a change in the geometry of the centra l 15,15'-cis bond of the spheroidenone. In addition, increased splitting of the nu(1) vibrational modes in two of the mutant Res 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 th e spheroidenone. These changes in the detailed conformation of the reaction centre carotenoid do not affect the optical properties of the cofactor, an d are beyond the limits of detection of X-ray crystallography as currently applied to the bacterial reaction centre.