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We report proposed chemical assignments of polarized nitrogen NMR signals from photosynthetic reaction centers of Rb. sphaeroides. These signals, which we previously described, are observed with solid state NMR methods in samples of Q-blocked reaction centers that are enriched in 15N. The CIDNP is excited by CW illumination with a Xenon arc lamp; we presume that they result from a radical pair mechanism (RPM) involving mixing of the electronic triplet and singlet spin states of P•+I•-. In this work selective labeling and comparison with chemical shifts of model compounds were used to assign the signals and were also used to distinguish directly and indirectly polarized signals. Signals at isotropic shifts of 163, 173, 232, and 236 ppm (relative to 1 M 15NH4Cl in 2 N HCl) were assigned as arising from the tetrapyrrole nitrogens of the special pair P865, and all appear to be directly polarized from the RPM. An additional small peak at 167 ppm appear to be another bacteriochlorophyll species, either the monomeric “B” or the “other half” of P865. Signals at 105, 113, and 276 ppm arise from the tetrapyrrole nitrogens of the bacteriopheophytin acceptor (“I”), and some of these signals (particularly the nitrogens in rings II and IV) seem to be directly polarized, while others are polarized by homonuclear spin diffusion involving a neighboring directly polarized nitrogen. Signals at 147 and 201 ppm arise from the δ and ε nitrogens of histidine, presumably from the ligand of P865, and are indirectly polarized. The intensities of the bacteriopheophytin signals are sensitive to the lifetime of 3P, consistent with a RPM mechanism in which 3P acts as the nuclear relaxant for I; this was concluded from comparisons of samples in which the acceptor QA was prereduced and samples in which is was chemically extracted, which are known from previous work to differ strongly in the lifetime of 3P. Many of the signals have chemical shift values that closely correspond to related model compounds, but moderate deviations (ca. 10 ppm) are seen for a few including the histidine resonances.

Solid-state NMR spectra of natural abundance 13C in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides R-26 was measured. When the quinone acceptors were removed and continuous visible illumination of the sample was provided, exceptionally strong nuclear spin polarization was observed in NMR lines with chemical shifts resembling those of the aromatic carbons in bacteriochlorophyll and bacteriopheophytin. The observation of spin polarized 15N nuclei in bacteriochlorophyll and bacteriopheophytin was previously demonstrated with nonspecifically 15N-labeled reaction centers. Both the carbon and the nitrogen NMR studies indicate that the polarization is developed on species that carry unpaired electrons in the early electron transfer steps, including the bacteriochlorophyll dimer donor P860 and probably the bacteriopheophytin acceptor I. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum; most lines were absorptive but the methine carbons of the porphyrin ring (a, .3, y, 8) exhibited emissive polarization. The change in the sign of the hyperfine coupling at these sites indicates the existence of nodes in the spin density distribution on the tetrapyrrole cofactors flanking each methine carbon bridge.We previously reported that light-induced nuclear spin polarization in the 15N solid-state NMR (SSNMR) spectra of photosynthetic reaction centers made it possible to detect nitrogen nuclei located in the active site of the protein with enormous intensities not attainable under typical experimental conditions (1). These studies were done using nonspecifically labeled reaction centers in which the quinone acceptors were either missing or chemically reduced; the known photochemical cycles of the remaining bacteriochlorophyll and bacteriopheophytin donor and acceptor species make them probable candidates for radical-pair-type chemically induced dynamic nuclear polarization (CIDNP) processes (2). A paper with the chemical assignments for the 15N polarized signals is forthcoming (3). We now report the observation of photo-CIDNP that allows us to detect the SSNMR spectra of natural abundance 13C in quinone-depleted photosynthetic reaction centers from Rhodobacter sphaeroides R-26 (see Fig. 1).The study of large biological systems by SSNMR usually requires specific isotopic labeling with an NMR observable nucleus, which can be burdensome and expensive in some cases and impossible in others. Specific isotopic labeling has been used in the study of photosynthetic reaction centers from R. sphaeroides (4, 5), as well as in other systems of biophysical interest (6). The finding we report is remarkable in the sense that exceptionally large NMR signals from the active site of a large protein are obtained even in the absence of any kind of isotopic enrichment.The reaction center from R. sphaeroides R-26 is a transmembrane protein with a known structure (7), consisting of three subunits (L, M, and H) and nine cofactors (four bacteriochlorophylls, two bacteriopheophytins, two qu...