Bacteriopheophytin a in the Active Branch of the Reaction Center of Rhodobacter sphaeroides Is Not Disturbed by the Protein Matrix as Shown by ¹³C Photo-CIDNP MAS NMR

Abstract

The electronic structure of bacteriopheophytin a (BPhe a), the primary electron acceptor (ΦA) in photosynthetic reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides, is investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) magic-angle spinning (MAS) NMR spectroscopy at atomic resolution. By using various isotope labeling systems, introduced by adding different (13)C selectively labeled δ-aminolevulinic acid precursors in the growing medium of R. sphaeroides wild type (WT), we were able to extract light-induced (13)C NMR signals originating from the primary electron acceptor. The assignments are backed by theoretical calculations. By comparison of these chemical shifts to those obtained from monomeric BPhe a in solution, it is demonstrated that ΦA in the active branch appears to be electronically close to free bacteriopheophytin. Hence, there is little effect of the protein surrounding on the cofactor functionally which contributes with its standard redox potential to the electron transfer process that is asymmetric.

Analysis of the Electronic Structure of the Special Pair of a Bacterial Photosynthetic Reaction Center by 13C Photochemically Induced Dynamic Nuclear Polarization Magic‐Angle Spinning NMR Using a Double‐Quantum Axis

“…The lineshape is more than 90% Lorentzian. That is in-line with previous experiments (35,81) concluding that the narrow line width is a strong argument for a structurally stiff and well structured cofactor arrangement along the electron transfer chain.…”

“…Analysis of the RCs of R. sphaeroides, employing photo-CIDNP MAS NMR, has demonstrated that the origin of the functional symmetry break is not due to the acceptor (35) but that the electronic structure is already broken in the "dark" ground-state electronic structure (36)(37)(38). Also in the radical-pair state, the electron spin density is asymmetrically distributed over the Special Pair (38), an observation well in-line with previous 1 H-ENDOR studies (39).…”

“…Hence, the ground‐state electronic symmetry is already broken . As reason for such symmetry break, not far‐reaching Coulomb effects, no difference on the acceptor site but local effects from side‐chain folding and assumable particular macrocycle foldings are responsible. On the primary electron acceptor cofactor, both upfield and downfield shifts of a negligible magnitude (∆ σ < 2.6) imply an electronic structure similar to that of a free bacteriopheophytin is acetone .…”

“…[42][43][44] and dipolar-assisted rotational resonance (DARR, see Ref. 35,41,45), have been adapted to the photo-CIDNP experiment by removal of the initial crosspolarization (CP) step. The removed CP step is primarily used as a strategy to enhance the sensitivity of low-gamma nuclei by transferring proton polarization.…”

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

“…showing the same features as observed in spectra of an u-13 C-ALA labeled sample under continuous illumination 21 . The slightly dispersive difference shape of the carbonyl signal probably originates from potential phase shifts by heating.…”