Role of the Protein Cavity in Phytochrome Chromoprotein Assembly and Double‐bond Isomerization: A Comparison with Model Compounds

Abstract

Difference patterns of (13)C NMR chemicals shifts for the protonation of a free model compound in organic solution, as reported in the literature (M. Stanek, K. Grubmayr [1998] Chem. Eur. J.4, 1653-1659), were compared with changes in the protonation state occurring during holophytochrome assembly from phycocyanobilin (PCB) and the apoprotein. Both processes induce identical changes in the NMR signals, indicating that the assembly process is linked to protonation of the chromophore, yielding a cationic cofactor in a heterogeneous, quasi-liquid protein environment. The identity of both difference patterns implies that the protonation of a model compound in solution causes a partial stretching of the geometry of the macrocycle as found in the protein. In fact, the similarity of the difference pattern within the bilin family for identical chemical transformations represents a basis for future theoretical analysis. On the other hand, the change of the (13)C NMR chemical shift pattern upon the Pr --> Pfr photoisomerization is very different to that of the free model compound upon ZZZ --> ZZE photoisomerization. Hence, the character of the double-bond isomerization in phytochrome is essentially different from that of a classical photoinduced double-bond isomerization, emphasizing the role of the protein environment in the modulation of this light-induced process.

Color Tuning in Red/Green Cyanobacteriochrome AnPixJ: Photoisomerization at C15 Causes an Excited-State Destabilization

“…For this reason, an A-ring rotation about C5 can be ruled out although one of its adjoining carbons, C6, exhibits a 5.0-ppm downfield shift that can be ascribed to the Pg deprotonation of the B-ring pyrrole nitrogen. 39 The state-induced protonation change of the AnPixJg2 chromophore when generated in vitro is also evidenced by the significant δ C change at C9 position (5.2 ppm). As shown with Raman spectroscopy, for in vivo generated AnPixJg2, all four pyrrole nitrogens are protonated in both states 40 as seen in phytochromes.…”

“…The persistence of the pattern points to the conservation of the same photochemical mechanism, occurring, however, with an inverted electronic effect. Since the photoisomerization of free bilins in organic solution shows an entirely different Δδ C pattern from that observed for both proteins, 39 we take this continuity as convincing evidence for a double-bond photoisomerization by the same mechanism. In the case of Cph1Δ2 (Figure 4a), red spheres are dominant in this green-shaded region, indicative of a decrease of local electron density for its 15E PCB as Pfr, whereas formation of the green-absorbing 15E species of AnPixJg2 (Figure 4b) leads to an increase of local electron density in the same region.…”

Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2‐Pr of Agrobacterium fabrum: Isomerization in a pH‐dependent H‐bond Network

“…Generally, twisting motions around the methine double bonds are expected to characterize the initial chromophore structural evolution after π‐electron photoexcitation. In fact, isomerization around the C5 and C15 double bond has been observed for free PCB and around the C5 or C15 but not the C10 double bond for free BV–dimethylester–IXa, whereas, caused by the constraints in the binding pocket, canonical phytochromes described so far exclusively exhibit C15=C16 double bond photoisomerization. Nevertheless, dependent on the specific protein environment, a partial involvement of the C5 torsional coordinate (or ring A conformation) in the Pr–Pfr phototransformation appears plausible, as for example, proposed for Cph1‐Pr ( Cyanobacterium Synechocystis ), for Synechococcus OS‐B′ phytochrome and for PaBphP .…”

“…Generally,twisting motions around the methine double bonds are expected to characterize the initial chromophores tructural evolution after p-electron photoexcitation. In fact, isomerization around the C5 [41] and C15 double bond has been observed for free PCB [65] anda roundt he C5 or C15 but not the C10 double bond for free BV-dimethylester-IXa, [65][66][67] whereas, caused by the constraints in the binding pocket, canonical phytochromes described so far exclusively exhibit C15=C16 double bond photoisomerization.N evertheless, dependent on the specific protein environment, ap artial involvement of the C5 torsional coordinate (or ring Ac onformation)i nt he Pr-Pfr phototransformationa ppears plausible, as for example, proposed for Cph1-Pr (Cyanobacterium Synechocystis), [26] for Synechococcus OS-B' phytochrome [68] and forP aBphP. [69] In the sequential scheme under discussion the reaction coordinate must finally merge into twist and then complete rotation around the C15=C16 bond in the reactive branch to lumi-R, and for the majority of the excited chromophores( the nonreactive branch to Pr) into the reversal of all previouss tructuralc hanges including the back-flipo fr ing D. Accordingly,t he stabilizationo fb oth the ring B/C andt he ring Dm oiety at pH 9( with respectt op H6) point to contributiono fC 5a nd C15 torsion to the reaction coordinate, respectively,a nd to the observed slowing down of the corresponding kinetics.…”

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome

“…1) and at the GAF/ PHY-tongue interface are highly conserved in phytochromes. [17][18][19] The collective interactions between these conserved residues as well as water molecules around the chromophore are critical for the phytochrome photochemistry, photoisomerization, and photoconversion. 14,[20][21][22] Among various conserved residues, the Y263 resides close to the chromophore at the GAF/PHY-tongue interface in bacterial phytochromes.…”