Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Variation of excitation energy influences the product distribution of a two-step electron transfer: S(2) vs S(1) electron transfer in a Zn(II)porphyrin-viologen complex.

We have, for the first time for molecular systems in solution, shown a case where the variation of excitation energy influences the product distribution of a two-step electron transfer. The photoexcitation was to the porphyrin-localized S(2) state or either of the S(1)(v = 1) or S(1)(v = 0) states of an aqueous Zn(II)-meso-tetrasulphonatophenyl-porphyrin-methylviologen (ZnTPPS(4-)/MV(2+)) complex. Both forward and back electron transfer occur on a subpicosecond time scale (tau(FET) approximately 0.2, tau(BET) = 0.7 ps). The excess energy of the higher excitations partially survives both electron transfer steps. This is seen by different distributions of unrelaxed ground states, which are generated by the back electron transfer and has unique UV-vis spectroscopic signatures. State selective electron transfer opens interesting possibilities for reaction control, and the results represent initial steps in that direction.

Solvent dependent structural perturbations of chemical reaction intermediates visualized by time-resolved x-ray diffraction.

Ultrafast time-resolved wide angle x-ray scattering from chemical reactions in solution has recently emerged as a powerful technique for determining the structural dynamics of transient photochemical species. Here we examine the structural evolution of photoexcited CH(2)I(2) in the nonpolar solvent cyclohexane and draw comparisons with a similar study in the polar solvent methanol. As with earlier spectroscopic studies, our data confirm a common initial reaction pathway in both solvents. After photoexcitation, CH(2)I(2) dissociates to form CH(2)I* + I*. Iodine radicals remaining within the solvent cage recombine with a nascent CH(2)I* radical to form the transient isomer CH(2)I-I, whereas those which escape the solvent cage ultimately combine to form I(2) in cyclohexane. Moreover, the transient isomer has a lifetime approximately 30 times longer in the nonpolar solvent. Of greater chemical significance is the property of time-resolved wide angle x-ray diffraction to accurately determine the structure of the of CH(2)I-I reaction intermediate. Thus we observe that the transient iodine-iodine bond is 0.07 A+/-0.04 A shorter in cyclohexane than in methanol. A longer iodine-iodine bond length for the intermediate arises in methanol due to favorable H-bond interaction with the polar solvent. These findings establish that time-resolved x-ray diffraction has sufficient sensitivity to enable solvent dependent structural perturbations of transient chemical species to be accurately resolved.

Ultrafast electron transfer dynamics of a Zn(II)porphyrin-viologen complex revisited: S2 vs S1 reactions and survival of excess excitation energy.

The photoinduced electron transfer reactions in a self-assembled 1:1 complex of zinc(II)tetrasulphonatophenylporphyrin (ZnTPPS(4-)) and methylviologen (MV(2+)) in aqueous solution were investigated with transient absorption spectroscopy. ZnTPPS(4-) was excited either in the Soret or one of the two Q-bands, corresponding to excitation into the S(2) and S(1) states, respectively. The resulting electron transfer to MV(2+) occurred, surprisingly, with the same time constant of τ(FET) = 180 fs from both electronic states. The subsequent back electron transfer was rapid, and the kinetics was independent of the initially excited state (τ(BET) = 700 fs). However, ground state reactants in a set of vibrationally excited states were observed. The amount of vibrationally excited ground states detected increased with increasing energy of the initial excited state, showing that excess excitation energy survived a two-step electron transfer reaction in solution. Differences in the ZnTPSS(•3-)/MV(•+) spectra suggest that the forward electron transfer from the S(2) state at least partially produces an electronically excited charge transfer state, which effectively suppresses the influence of the inverted regime. Other possible reasons for the similar electron transfer rates for the different excited states are also discussed.

Light-induced structural changes in a photosynthetic reaction center caught by Laue diffraction.

Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

Structural dynamics of light-driven proton pumps.

Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.