Phototropin is a flavin mononucleotide (FMN) containing blue-light receptor, which regulates, governed by its two LOV domains, the phototropic response of higher plants. Upon photoexcitation, the FMN cofactor triplet state, 3F, reacts with a nearby cysteine to form a covalent adduct. Cysteine-to-alanine mutants of LOV domains instead generate a flavin radical upon illumination. Here, we explore the formation of photochemically induced dynamic nuclear polarization (CIDNP) in LOV2-C450A of Avena sativa phototropin and demonstrate that photo-CIDNP observed in solution 13C NMR spectra can reliably be interpreted in terms of solid-state mechanisms including a novel triplet mechanism. To minimize cross-polarization, which transfers light-induced magnetization to adjacent 13C nuclei, our experiments were performed on proteins reconstituted with specifically 13C-labeled flavins. Two potential sources for photo-CIDNP can be identified: The photogenerated triplet state, 3F, and the triplet radical pair 3(F–•W+•), formed by electron abstraction of 3F from tryptophan W491. To separate the two contributions, photo-CIDNP studies were performed at four different magnetic fields ranging from 4.7 to 11.8 T. Analysis revealed that, at fields <9 T, both 3(F–•W+•) and 3F contribute to photo-CIDNP, whereas at high magnetic fields, the calculated enhancement factors of 3F agree favorably with their experimental counterparts. Thus, we have for the first time detected that a triplet state is the major source for photo-CIDNP in a photoactive protein. Since triplet states are frequently encountered upon photoexcitation of flavoproteins, the novel triplet mechanism opens up new means of studying electronic structures of the active cofactors in these proteins at atomic resolution.

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700+A1A– and P700+A1B– of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700+ in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc.2001, 123, 4211–4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B– on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700+A1A– and P700+A1B– participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700+A1A–. A detailed comparison reveals different orientations of A1A– and A1B– in their respective binding sites such that formation of a strong hydrogen bond from A1– to the protein backbone is possible only in the case of A1A–. We suggest that this is relevant to the rates of forward electron transfer from A1A– or A1B– to the iron–sulfur center FX, which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.

The unique physical properties of photoexcited triplet states have been explored in numerous spectroscopic studies employing electron paramagnetic resonance (EPR). So far, however, no quantum interference effects were found in these systems in the presence of a magnetic field. In this study, we report the successful EPR detection of nuclear quantum oscillations in an organic triplet state subject to an external magnetic field. The observed quantum coherences can be rationalized using an analytical theory. Analysis suggests that the nuclear spins are actively involved in the intersystem crossing process. The novel mechanism also acts as a source of oscillatory nuclear spin polarization that gives rise to large signal enhancement in nuclear magnetic resonance (NMR). This opens new perspectives for the analysis of chemically induced dynamic nuclear polarization in mechanistic studies of photoactive proteins.

Using the density operator formalism, an analytical model is developed to study the time evolution of the electron spin magnetization of photo-excited triplet states in an external magnetic field. Analysis reveals that pulsed light excitation initiates an oscillatory electron spin magnetization in the direction of the external field. The frequency of the quantum oscillations is determined by the electron Zeeman and dipolar interactions. The oscillation amplitude reaches a maximum when the electron Zeeman splitting matches the energy of a zero-field transition of the triplet state. This suggests that the predicted quantum oscillations can be detected only at low magnetic fields.Quantum oscillations are induced in the electron spin magnetization of photo-excited triplet states in an external magnetic field.