Gudrun Hermann

Protochlorophyllide a: A Comprehensive Photophysical Picture

The photochemistry of protochlorophyllide a, a precursor in the biosynthesis of chlorophyll and substrate of the light regulated enzyme protochlorophyllide oxidoreductase, is investigated by pump-probe spectroscopy. Upon excitation into the lowest lying Q-band the light induced changes are recorded over a wide range of probe wavelengths in the visible and near-IR region between 500 and 1000 nm. Following excitation, an initial ultrafast 450 fs process is observed related to the motion out of the Franck-Condon region on the excited state surface; thus directly unraveling previous suggestions based on time-resolved fluorescence measurements (ChemPhysChem 2006, 7, 1727-1733). Furthermore, the data reveals a previously concealed photointermediate, whose formation on a nanosecond timescale matches the overall fluorescence decay and is assigned to a triplet state. The implications of this finding with respect to the photochemistry of NADPH:protochlorophyllide oxidoreductase (POR) are discussed.

Picosecond Absorption and Fluorescence Studies on Large Phytochrome from Rye

Summary For the first time, picosecond absorption and fluorescence spectroscopy have been jointly applied to the study of the primary photoprocesses in large phytochrome. It is shown that the excited state lifetime in the red absorbing form of phytochrome is ∼40 ps, whereas lumi-R known hitherto as the primary photoproduct is formed only within ∼100 ps. This discrepancy suggests that lumi-R is not directly formed from excited state PR, but there exists a hitherto unknown intermediate which we have designated prelumi-R.

Femtosecond Spectroscopic Studies on the Red Light-Absorbing Form of Oat Phytochrome and 2,3-Dihydrobiliverdin

Abstract— The excited state behavior of the red light‐absorbing form of phytochrome (Pr) was studied on the femtosecond time scale. After excitation of Pr with 75 fs laser pulses at 616 nm the kinetics of the transient absorption changes was recorded at selected wavelengths probing mainly the bleaching of the Pr ground‐state absorption and the stimulated emission. The kinetic data obtained indicate the population of an excited state with a 3 ps lifetime immediately after excitation. This state precedes the formation of another excited state with a 32 ps lifetime. The decay of the latter state is followed by the appearance of a first product state that is assumed to represent lunii‐R. In addition, 2,3‐dihydrobiliverdin, which is considered to be an adequate model of the Pr chro‐mophore, was included in the femtosecond studies. The absorption difference spectra recorded at various delay times show an immediate bleaching of the ground‐state absorption. Simultaneously with bleaching a broad transient absorption appears between 410 and 525 nm. The data analysis yields similar kinetic components as they were observed in the decay of Pr. It is suggested from this finding that within the first tens of picoseconds after excitation the excited‐state properties of Pr are mainly determined by the properties of the chromophore itself.

PICOSECOND DYNAMICS OF THE EXCITED STATE RELAXATIONS IN PHYTOCHROME

Abstract— A comparative study of the picosecond kinetics of rye (Secale cereale L.) phytochrome, its 39 and 23 kDa chromopeptides and deuterated rye phytochrome has been carried out. Evidence is presented that the fluorescence decay of Pr contains a very short lifetime component (14 ps) which has escaped detection in the fluorescence studies reported so far. Thus, the overall decay is well described by four exponential components, two rapid (14 and 44 ps) and two slower ones (157 and 690 ps). The fluorescence decays of deuterated Pr and of a 39 and 23 kDa chromopeptide of Pr also require the analysis in terms of four exponentials for a good fit. Some of the lifetime and amplitude values obtained differ significantly from the values estimated for Pr. In the chromopeptides, the two longer components have distinctly slower decays. For the two faster components the lifetimes remain approximately the same, but their relative amplitudes vary greatly. In deuterated Pr, the lifetimes are affected only slightly by deuteration. In contrast, the decay amplitudes are strikingly altered. Moreover, from a rate equation simulation modelling the observed fluorescence kinetics, it turns out that the yields for the various deactivation steps in the chromopeptides and in deuterated Pr reveal differences from the corresponding values in Pr. The implications of the results presented with respect to the influence of the protein moiety of Pr on the picosecond relaxation process are discussed.

Conformational flexibility of phycocyanobilin: An AM1 semiempirical study

Semiempirical AM1 studies on phycocyanobilin, the most abundant chromophore in the light-harvesting phycobiliproteins and a suitable model of the phytochrome chromophore, have been carried out. For the all-Z isomer, the structures and energies of all possible conformers, which arise from rotation about the single bonds of the exocyclic methine bridges, were calculated. In addition, the rotational barriers between the different conformers were deduced from the computed energy hypersurfaces. The cyclic helical SSS conformation is the most stable structure. Other minimum-energy structures with well-separated local minima on the energy hypersurface are the partially extended ASS and SAS conformations, although the conformational situation is characterized by a high degree of kinetic flexibility. The energy of the different conformers is mainly governed by intramolecular hydrogen bonding and steric repulsion of the substituents at the tetrapyrrolic backbone. It is very likely that the energetically most favorable conformers, SSS, ASS, and SAS, correspond with the three ground-state conformers of phycocyanobilin detected in previous time-resolved absorption studies.

Picosecond events in the phototransformation of phytochrome - a time-resolved absorption study?

Abstract Picosecond time-resolved transient absorption studies with phytochrome in its red light absorbing form (P r ) isolated from rye ( Secale cereale cv. Danae ) are reported. After excitation of P r with 6 ps pulses at 621 nm the transient absorption spectra were measured at various delay times from 5 ps up to 1 ns. Upon excitation an increase in the transient absorption around 400 nm immediately appeared, which is ascribed to the first excited state absorption of P r . With a delay of about 15 ps a strong bleaching of the P r ground state absorption around 665 nm emerged. After a series of further absorption changes, an increase in absorbance occurred above 685 nm. Since this transient absorption persisted over the 1 ns time-window measured, it is interpreted as being due to the formation of lumi-R. Based on the time evolution of the observed absorption changes it seems likely that two pools of lumi-R were formed. In addition, it was found from the kinetics of the picosecond absorption changes that the relaxation of excited P r obviously involves processes which follow a non-exponential rate law. From a comparison with picosecond adsorption spectroscopy on deuterated P r and a 39 kDa chromopeptide, conclusions about the first relaxation steps in the phototransformation of P r are drawn. It is suggested that they involve rotations at the single bonds in the chromophoric methine bridges.

Protein-Induced Excited-State Dynamics of Protochlorophyllide

The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. PORs unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.

Catalytic Efficiency of a Photoenzyme—An Adaptation to Natural Light Conditions

During evolution nature has developed several photoreceptors with special chromophoric groups to absorb visible light, which is subsequently used as a source of energy or bit of information to control biological processes. 2] The underlying light-induced processes take place on very short time scales (picoseconds) whereby possible loss processes are minimized and the efficiency of the biological signal, generating the primary process, is increased. A central problem of current research consists in elucidating the molecular processes of the evolutionary optimized biological reactions in order to design biomimetic systems of similar efficiency. In this respect exceptional progress has been made within the last years. For example, beside understanding the primary processes of photosynthesis, the ultrafast reaction mechanism of the isomerization of 11-cis-retinal in rhodopsin, the Z,E isomerization of the bilin chromophore in phytochrome, or the repair of UV-light induced DNA damage have been elucidated. Particular interest consists in unraveling the molecular dynamics of enzyme-catalyzed reactions in order to understand the reason for the high selectivity and efficiency of enzyme catalysis. Photoenzymes, whose catalytic activity is initiated by the absorption of a photon, are particularly well suited for such investigations. They provide the unique possibility to monitor the reaction process in real time by time-resolved spectroscopy. There are only two enzymes in nature, which require light for their catalytic activity. One is DNA photolyase and the other one is NADPH:protochlorophyllide oxidoreductase (POR). POR is a key enzyme of the chlorophyll biosynthesis in all organisms showing oxygenic photosynthesis. In one of the last steps of the chlorophyll biosynthesis POR reduces protochlorophyllide (PChlide) to chlorophyllide (Chlide) upon absorption of light and in the presence of the cofactor NADPH (see Figure 1 A). In the course of the photoreaction a coupled H /H transfer onto the C(17)=C(18) bond of PChlide occurs,

Excited state relaxations of phytochrome studied by femtosecond spectroscopy

Abstract Femtosecond time-resolved spectroscopic studies with phytochrome in its red light absorbing form (Pr) were performed. Upon excitation of Pr with 75 fs pulses at 618 nm bleaching of the Pr ground state absorption accompanied by a broad transient absorption centered near 725 nm was observed. The stimulated emission of Pr was detected with a delay of about 200 fs. The appearance of stimulated emission coincided with a strong decrease of the transient absorption near 725 nm. From these results it is suggested that Pr is initially excited into a vibrationally unrelaxed state that undergoes relaxation within about 200 fs towards the fluorescing state. From 1 ps after excitation the initially fast relaxation processes were followed by comparatively slower ones. From decay kinetics of bleaching and fluorescence in the first 6 ps after excitation a decay time of about 15 ps was estimated.

Flourescence Lifetime of the Information Processing Plant Pigment Phytochrome

Summary Fluorescence lifetime of buffered aqueous solutions of large phytochrome has been determined at room temperature using a laser phase fluorometer with subnanosecond resolution. By studying the decay characteristics in dependence on the wavelength of the fluorescence the decay time was found to decrease with increasing wavelength over the fluorescence band. At the wavelength of the fluorescence maximum (λ = 678 nm) and the modulation frequency of 78.4 MHz the effective lifetime was determined to be 0.4 us. Measurements of the effective fluorescence lifetime at different modulation frequencies indicate that the fluorescence decay is not single exponential.