13C magic angle spinning NMR study of the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in [4'-13C]tyrosine.

Solid-state 13C magic angle spinning (MAS) NMR has been used to investigate detergent-solubilized photosynthetic reaction centers of Rhodobacter sphaeroides R26, selectively enriched in [4-13C]-tyrosine. The reaction centers were frozen, in the dark and while subject to intense illumination, and studied at temperatures between approximately 215 and approximately 260 K. The signal consists of at least seven narrow lines superimposed on a broad doublet. The chemical shift anisotropy is similar to that for crystalline tyrosine. The two narrowest resonances, corresponding to signals from individual tyrosines, are 28 +/- 5 Hz wide, comparable to what is observed for quaternary carbons in linearly elastic organic solids. The line width as well as the chemical shift of these signals is essentially independent of temperature. This provides strong evidence for an unusually ordered, well-shielded, and structurally, electrostatically, and thermodynamically stable interior of the protein complex without structural heterogeneities. As the temperature is lowered, additional signal from the labels develops and the natural abundance resonances from the detergent broaden, providing evidence for considerable flexibility at the exterior of the protein complex and in the detergent belt at the higher temperatures. In addition, the NMR provides evidence for an electrostatically uniform and neutral complex, since the total dispersion in isotropic shifts for the labels is < 5 ppm and corresponds to electron density variations of less than 0.03 electronic equivalents with respect to tyrosine in the solid state or in solution. When the sample is frozen while subject to intense illumination, a substantial part of the protein is brought into the charge-separated state P.+QA.-. At least three sharp resonances, including the narrowest lines, are substantially reduced in intensity. It is argued that this effect is caused by the electronic spin density associated with the oxidized primary donor P.+. These results strongly suggest that the environment of the special pair is extremely rigid and question the role of protein conformational distortions during the primary photoprocess.