Quinone (QB) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

• Published in: Biochemistry (Easton) 2003-05, Vol.42 (19), p.5819-5827
• Main Authors: Nabedryk, Eliane, Breton, Jacques, Sebban, Pierre, Baciou, Laura
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Binding Sites - genetics; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - genetics; Photosynthetic Reaction Center Complex Proteins - metabolism; Photosynthetic Reaction Center Complex Proteins - radiation effects; Quinones - chemistry; Quinones - metabolism; Rhodobacter sphaeroides - genetics; Rhodobacter sphaeroides - metabolism; Rhodobacter sphaeroides - radiation effects; Spectroscopy, Fourier Transform Infrared
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi034240d

The effect of substituting Pro-L209 with Tyr, Phe, Glu, and Thr in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was investigated by monitoring the light-induced FTIR absorption changes associated with the photoreduction of the secondary quinone QB. Pro-L209 is close to a chain of ordered water molecules connecting QB to the bulk phase. In wild-type RCs, two distinct main QB binding sites (distal and proximal to the non-heme iron) have been described in the literature. The X-ray structures of the mutant RCs Pro-L209 → Tyr, Pro-L209 → Phe, and Pro-L209 → Glu have revealed that QB occupies a proximal, intermediate, and distal position, respectively [Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G. (2001) Biochemistry 40, 4253−4260]. FTIR absorption changes associated with the reduction of QB in Pro-L209 → Phe RCs reconstituted with 13C-labeled ubiquinone show a highly specific IR fingerprint for the CO and CC modes of QB upon selective labeling at C1 or C4. This IR fingerprint is similar to those of wild-type RCs and the Pro-L209 → Tyr mutant [Breton, J., Boullais, C., Mioskowski, C., Sebban, P., Baciou, L., and Nabedryk, E. (2002) Biochemistry 41, 12921−12927], demonstrating that equivalent interactions occur between neutral QB and the protein in wild-type and mutant RCs. It is concluded that in all RCs, neutral QB in its functional state occupies a unique binding site which is favored to be the proximal site. This result contrasts with the multiple QB binding sites found in crystal structures. With respect to wild-type RCs, the largest FTIR spectral changes upon QB - formation are observed for the Phe-L209 and Tyr-L209 mutants which undergo similar protein structural changes and perturbations of the semiquinone modes. Smaller changes are observed for the Glu-L209 mutant, while the vibrational properties of the Thr-L209 mutant are essentially the same as those for native RCs.