Bilin-binding photoreceptors are light-signaling proteins that mediate various processes from photomorphogenesis, phototaxis, chromatic acclimation, to photosynthesis. They are also promising tunable optical agents for use in optogenetics and superresolution microscopy. Using an integrated approach of crystallography, spectroscopy, and QM/MM calculations, this work examines the ultrafast dynamics of a photoactive single-domain phytochrome. Our work reveals in detail the critical role of the protein environment in defining the excited state lifetime and thereby the quantum efficiency of the bilin photoisomerization. This insight provides design principles for engineering of bilin-based photoreceptors for biotechnological and medical applications.Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.
Two covalently linked donor–acceptor copper phenanthroline complexes (C–A dyads) of interest for solar energy conversion/storage schemes, [Cu(i)(Rphen(OMV)24+)2]9+ = RC+A48+ with RC+ = [Cu(i)Rphen2]+ involving 2,9-methyl (R = Me) or 2,9-phenyl (R = Ph)-phenanthroline ligands that are 5,6-disubstituted by 4-(n-butoxy) linked methylviologen electron acceptor groups (A2+ = OMV2+), have been synthesized and investigated via quantum chemical calculations and nanosecond laser flash spectroscopy in 1,2-difluorobenzene/methanol (dfb/MeOH) mixtures. Upon photoexcitation, charge transfer (CT) states RC2+A+A36+ are formed in less than one ns and decay by charge recombination on a time scale of 6–45 ns. The CT lifetime of RC2+A+A36+ has a strong dependence on MeOH solvent fraction when R = Me, but is unaffected if R = Ph. This solvent effect is due to coordination of MeOH solvent in MeC+A48+ (i.e. exciplex formation) allowed by conformational flattening of the ligand sphere, which cannot occur in PhC+A48+ having bulkier Phphen ligand framework. Interestingly, the decay time of the CT state increases for both species at low magnetic fields with a maximum increase of ca. 30% at ca. 150 mT, then decreases as the field is increased up to 1500 mT, the highest field investigated. This magnetic field effect (MFE) is due to magnetic modulation of the spin dynamics interconverting 3CT and 1CT states. A quantitative modeling according to the radical pair mechanism involving ab initio multireference calculations of the complexes revealed that the spin process is dominated by the effect of Cu hyperfine coupling. The external magnetic field suppresses the hyperfine coupling induced spin state mixing thereby lengthening the CT decay time. This effect is counteracted by the field dependent processes of T0–S mixing through the Δg-mechanism and by a local mode spin–orbit mechanism. Further, the maximum MFE is limited by a finite rate of direct recombination of 3CT states and the spin-rotational mechanism of spin relaxation. This study provides a first comprehensive characterization of Cu(ii)-complex spin chemistry and highlights how spin chemistry can be used to manipulate solar energy harvesting and storage materials.
Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.