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

Abstract

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.

15 N photochemically induced dynamic nuclear polarization magic-angle spinning NMR analysis of the electron donor of photosystem II

“…Both the two emissive as well as the enhanced absorptive set of signals can be assigned conveniently to Chl cofactors (Table 1) based on assignment obtained on Chls in solution (19). At the donor site, which has been shown to be very rigid (15,20), slow signal recovery is expected. Hence, an experiment with very short cycle delay of 0.4 s (Fig.…”

“…Two more signals appear at 295.0 and 138.3 ppm, which can be conveniently assigned to N-IV and N-III of the primary electron acceptor, a Phe cofactor. The donor signals are remarkably narrow [full width at half-height (FWHH) of Ϸ40 Hz], whereas the acceptor signals are slightly broader (Ϸ70 Hz), indicating a general feature of photosynthetic RCs having a rigid donor site without structural heterogenities (15,20) and more structural flexibility at the acceptor site.…”

Photo-CIDNP in the Reaction Center of the Diatom Cyclotella meneghiniana Observed by 13C MAS NMR

“…The temperature was decreased slowly to ensure homogeneous sample distribution against the rotor wall. [54] Light and dark spectra were acquired using a Hahn-echo pulse sequence with two-pulse phase modulation (TPPM) proton decoupling. [55] 13 C MAS NMR spectra were collected at a temperature of 250 K with a deviation of approximately 2 K and under continuous illumination with white light using a 1000-W xenon-arc lamp.…”

Photochemically Induced Dynamic Nuclear Polarization Observed by Solid-State NMR in a Uniformly 13C-Isotope-Labeled Photosynthetic Reaction Center

“…The sample was loaded into a clear 4-mm sapphire rotor and inserted into the MAS probe. The sample was frozen at a low spinning frequency of 1400 Hz to ensure a homogeneous sample distribution 22 . The light and dark spectra were collected using a Hahn echo pulse sequence with the CYCLOPS phase cycle of the (π/2) pulse.…”