Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash C Photo-CIDNP MAS NMR

Abstract

Photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance (photo-CIDNP MAS NMR) allows for the investigation of the electronic structure of the photochemical machinery of photosynthetic reaction centers (RCs) at atomic resolution. For such experiments, either continuous radiation from white xenon lamps or green laser pulses are applied to optically dense samples. In order to explore their optical properties, optically thick samples of isolated and quinone-removed RCs of the purple bacteria of Rhodobacter sphaeroides wild type are studied by nanosecond laser-flash (13)C photo-CIDNP MAS NMR using excitation wavelengths between 720 and 940 nm. Action spectra of both the transient nuclear polarization as well as the nuclear hyperpolarization, remaining in the electronic ground state at the end of the photocycle, are obtained. It is shown that the signal intensity is limited by the amount of accessible RCs and that the different mechanisms of the photo-CIDNP production rely on the same photophysical origin, which is the photocycle induced by one single photon.

Fig. 1

Structural arrangement of cofactors in the RCs of Rb. sphaeroides WT. Cofactors involved into the spin dynamics of the primary radical-pair formation are: the primary electron donor P is formed by two bacteriochlorophyll a molecules, P_L (right) and P_M (left), the (primary) electron acceptor Φ, a bacteriopheophytin a in the active L branch, and the carotenoid (Car). Other cofactors are shown in light grey (Protein Data Bank entry 1MX3)

Fig. 2

Kinetics and spin dynamics of electron transport in quinone-depleted RCs of Rb. sphaeroides WT. After absorption of a photon, from the photochemically excited state of the primary donor P*, an electron is transferred to the primary acceptor Φ, a bacteriopheophytin cofactor. This initial singlet radical pair ¹(P^·+Φ^·−) is in a nonstationary state and highly electron polarized. An electron back-transfer leads to the electronic ground state (dashed arrow). Due to hyperfine interaction with nuclei, the singlet state of the radical pair evolves also into a triplet state ³(P^·+Φ^·−). Concomitant to this process of spin intersystem crossing (ISC), electron polarization is transferred to nuclei by the TSM and by the DD mechanisms. Net nuclear polarization is created by unbalancing the decay pathways of the singlet and the triplet radical pair (singlet and triplet branches). In addition, in time-resolved experiments TNP can be observed directly from the singlet decay channel because in the triplet decay pathway (triplet branch, dotted arrow), paramagnetic interaction of the triplet state of the nearby carotenoid, having a lifetime of 5 till 10 μs, leads to transiently obscured polarization (TOP)

Fig. 3

Bacteriochlorophyll a (BChl) molecule with isotope label pattern biosynthetically introduced by feeding with 4-¹³C-δ-aminolevulinic acid (4-ALA). The bacteriopheophytin a (BPhe) cofactors are labeled according to the same pattern

Fig. 4

The solid line is an ultraviolet–visible-NIR spectrum of purified RCs of Rhodobacter (Rb.) sphaeroides WT obtained at room temperature. The spectrum is normalized to the highest signal intensity at 860 nm (left scale). The peak at 860 nm is assigned to the bacteriochlorophylls of P. The peaks at 803 and 756 nm are assigned to the accessory bacteriochlorophylls and the bacteriopheophytins, respectively. The dotted line is the action spectrum of photo-CIDNP intensities of C-19 of P_L (159.8 ppm) obtained 100 μs after the laser flash and plotted as a function of the wavelength. The action spectrum is normalized to the highest signal intensity obtained with an excitation wavelength at 860 nm (right scale)

Fig. 5

¹³C photo-CIDNP MAS NMR spectra of RCs of 4-ALA ¹³C-isotope labeled Rb. sphaeroides WT collected using laser pulses at wavelengths of 940 nm (A), 860 nm (B), 820 nm (C), 803 nm (D), 780 nm (E), 756 nm (F), 720 nm (G). The absorption maxima of BPhe correspond to trace F, of the accessory BChls to trace D and of the donor BChls to trace B. All the spectra have been collected at a magnetic field of 4.7 T and a temperature of 233 K. The laser pulse length was 8 ns and the laser energy was 28 mJ in all experiments

Fig. 6

¹³C photo-CIDNP MAS NMR spectra of RCs of 4-ALA ¹³C-isotope labeled Rb. sphaeroides WT collected using laser pulses at wavelengths of 860 nm (A), 803 nm (B) and 756 nm (C), respectively. The absorption maxima of the donor BChls correspond to trace A, of the accessory BChls to trace B and of the BPhe to trace C. Traces A, B and C show the superposition of three spectra having different time delays between the light pulse and the NMR detection pulse (black 0 μs, grey 10 μs, and light grey 100 μs). All the spectra have been collected at a magnetic field of 4.7 T and a temperature of 233 K. The laser pulse length was 8 ns and the laser power was 28.2 mJ in all experiments