Harald Otto

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

delta pH-induced fluorescence quenching of 9-aminoacridine in lipid vesicles is due to excimer formation at the membrane

The fluorescence of 9-aminoacridine (9-AA) is quenched in vesicular suspensions containing negatively charged lipid headgroups (e.g., phosphatidylserine) upon imposition of a transmembrane (inside acidic) pH-gradient. It is shown that this fluorescence loss is accompanied by the formation of 9-AA dimers that undergo a transition in the dimer excited state to a dimer-excimer state. This result has been obtained on the basis of the specific dimer fluorescence excitation and hypochromic absorbance spectra that are redshifted by maximally 275 cm-1 (4.4 nm) with respect to the corresponding monomer spectra, as well as by the detection of the characteristic broad excimer emission band, centered at 560 nm. The existence of the spectrally distinct dimer-excimer is further corroborated by fluorescence life-time measurements that indicate an increased lifetime of up to 24 ns for this complex as compared with the normal monomer fluorescence lifetime of 16 ns. The formation of this dimer-excimer complex from the monomers can be reversed completely and the original monomeric spectral properties restored after the abolishment of the electrochemical proton gradient. In addition to the delta pH-induced dimer redshift in absorbance and fluorescence excitation, a further small redshift in monomer absorbance, fluorescence excitation, and emission spectra is observed due solely to the presence of the negatively charged phospholipid headgroups.

The dynamics of adiabatic photoreactions as studied by means of the time structure of synchrotron radiation

Abstract The decay of the dual fluorescence of the para-substituted dialkylanilines (esters and nitriles) as well as the fluorescence decay of several triphenylmethane (TPM) dyes is investigated. The esters relax several times faster than the nitriles, and in the TPM series, relaxation is accelerated by increasing the substituent acceptor strength. In both cases, non-exponential decay behaviour is observed. It is more pronounced for the compounds with stronger driving forces along the reaction pathways. Freezing of the relaxation process transforms the decay into a mono-exponential one. This is discussed in terms of two models, namely by a log-gaussian distribution of relaxation times, and by a non-radiative sink model.

Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1

The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs‐biliverdin in which the C4 C5 and C5–C6 bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1‐5Zs the photoconversion proceeds via the Lumi‐R intermediate to Meta‐RA, but the following millisecond‐transition to Meta‐RC is blocked. Consistently, no transient proton release was detected. The photoconversion of Agp1‐5Zs is apparently arrested in a Meta‐RA‐like intermediate, since the subsequent syn to anti rotation around the C5–C6 bond is prevented by the lock. The Meta‐RA‐like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.

Between the ground- and M-state of bacteriorhodopsin the retinal transition dipole moment tilts out of the plane of the membrane by only 3°

The orientation of the transition dipole moments in the ground state and the M‐intermediate of bacteriorhodopsin were determined by time‐resolved and steady‐state polarized absorption spectroscopy on samples of oriented immobilized purple membranes. The angle between the transition dipole moment and the membrane normal decreases from 66.8±0.5° in the all‐trans ground state to 64.1±0.8° in the 13‐cis M‐state. The light‐induced isomerization of the chromophore is thus accompanied by an orientational change of only about 3° out of the plane of the membrane. The absorption anisotropy at 410 nm remains constant over more than 4 decades of time covering both the rise and decay of M. Conformational changes accompanying a sequential M1→M2 transition thus do not affect the chromophore orientation.

Strong Hydrogen Bond between Glutamic Acid 46 and Chromophore Leads to the Intermediate Spectral Form and Excited State Proton Transfer in the Y42F Mutant of the Photoreceptor Photoactive Yellow Protein

In the Y42F mutant of photoactive yellow protein (PYP) the photoreceptor is in an equilibrium between two dark states, the yellow and intermediate spectral forms, absorbing at 457 and 390 nm, respectively. The nature of this equilibrium and the light-induced protonation and structural changes in the two spectral forms were characterized by transient absorption, fluorescence, FTIR, and pH indicator dye experiments. In the yellow form, the oxygen of the deprotonated p-hydroxycinnamoyl chromophore is linked by a strong low-barrier hydrogen bond to the protonated carboxyl group of Glu46 and by a weaker one to Thr50. Using FTIR, we find that the band due to the carbonyl of the protonated side chain of Glu46 is shifted from 1736 cm(-1) in wild type to 1724 cm(-1) in the yellow form of Y42F, implying a stronger hydrogen bond with the deprotonated chromophore in Y42F. The FTIR data suggest moreover that in the intermediate spectral form the chromophore is protonated and Glu46 deprotonated. Flash spectroscopy (50 ns-10 s) shows that the photocycles of the two forms are essentially the same except for a transition around 5 mus that has opposite signs in the two forms and is due to the chemical relaxation between the two dark states. The two cycles are coupled, likely by excited state proton transfer. The Y42F cycle differs from wild type by the occurrence of a new intermediate with protonated chromophore between the usual I(1) and I(2) intermediates which we call I(1)H (370 nm). Transient fluorescence measurements indicate that in I(1)H the chromophore retains the orientation it had in I(1). Transient proton uptake occurs with a time constant of 230 mus and a stoichiometry of 1. No proton uptake was associated however with the formation of the I(1)H intermediate and the relaxation of the yellow/intermediate equilibrium. These protonation changes of the chromophore thus occur intramolecularly. The chromophore-Glu46 hydrogen bond in Y42F is shorter than in wild type, since the adjacent chromophore-Y42 hydrogen bond is replaced by a longer one with Thr50. This facilitates proton transfer from Glu46 to the chromophore in the dark by lowering the barrier, leading to the protonation equilibrium and causing the rapid light-induced proton transfer which couples the cycles.

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy.

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Chromophore reorientation during the photocycle of bacteriorhodopsin: experimental methods and functional significance

Light-induced isomerization leads to orientational changes of the retinylidene chromophore of bacteriorhodopsin in its binding pocket. The chromophore reorientation has been characterized by the following methods: polarized absorption spectroscopy in the visible, UV and IR; polarized resonance Raman scattering; solid-state deuterium nuclear magnetic resonance; neutron and X-ray diffraction. Most of these experiments were performed at low temperatures with bacteriorhodopsin trapped in one or a mixture of intermediates. Time-resolved measurements at room temperature with bacteriorhodopsin in aqueous suspension can currently only be carried out with transient polarized absorption spectroscopy in the visible. The results obtained to date for the initial state and the K, L and M intermediates are presented and discussed. The most extensive data are available for the M intermediate, which plays an essential role in the function of bacteriorhodopsin. For this intermediate the various methods lead to a consistent picture: the curved all-trans polyene chain in the initial state straightens out in the M intermediate (13-cis) and the chain segment between C(5) and C(13) tilts upwards in the direction of the cytoplasmic surface. The kink at C(13) allows the positions of beta-ionone ring and Schiff base nitrogen to remain approximately fixed.

Model Systems for the Investigation of the Opsin Shift in Bacteriorhodopsin

Donor-acceptor substituted styrenes and phenylbutadienes with substituents varying in donor and acceptor strength and as reconstituted chromophore-protein complexes were investigated as model compounds for the protonated Schiff base chromophore in bacteriorhodopsin (bR) both experimentally and theoretically. Charge distribution, donor-acceptor strength, and the shift of the absorption energy are correlated. The effect of the external electrostatic field was tested with a compound carrying an additional nonconjugated charge. The concept of overpolarization by the external charge, that is, the reversal of the relative importance of the two main resonance structures in S(0) and S(1), has been emphasized and related to a simple qualitative 2 x 2 interaction model. The variable donor approach is a new way for a better understanding of the Opsin shift in Bacteriorhodopsin.

Photoselection and transient linear dichroism with oriented immobilized purple membranes : evidence for motion of the C(20)-methyl group of the chromophore towards the cytoplasmic side of the membrane

Abstract— We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time‐resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time‐resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the M‐intermediate, the transition dipole moment tilts out of the plane of the membrane by about 3°. On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11° tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 Å and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M‐intermediate.