Phytochrome as molecular machine: revealing chromophore action during the Pfr --> Pr photoconversion by magic-angle spinning NMR spectroscopy.

The cyanobacterial phytochrome Cph1 can be photoconverted between two thermally stable states, Pr and Pfr. The photochemically induced Pfr --> Pr back-reaction has been followed at low temperature by magic-angle spinning (MAS) NMR spectroscopy, allowing two intermediates, Lumi-F and Meta-F, to be trapped. Employing uniformly (13)C- and (15)N-labeled open-chain tetrapyrrole chromophores, all four states-Pfr, Lumi-F, Meta-F, and Pr-have been structurally characterized. In the first step, the double bond photoisomerization forming Lumi-F occurs. The second step, the transformation to Meta-F, is driven by the release of the mechanical tension. This process leads to the break of the hydrogen bond of the ring D nitrogen to Asp-207 and triggers signaling. The third step is protonically driven allowing the hydrogen-bonding interaction of the ring D nitrogen to be restored. Compared to the forward reaction, the order of events is changed, probably caused by the different properties of the hydrogen bonding partners of N24, leading to the directionality of the photocycle.

The role of the chromophore in the biological photoreceptor phytochrome: an approach using chemically synthesized tetrapyrroles.

In plants and bacteria, phytochromes serve as light-inducible, red-/far-red light sensitive photoreceptors that control a wide range of photomorphogenetic processes. Phytochromes comprise a protein moiety and a covalently bound bilin chromophore. Bilins are open-chain tetrapyrrole compounds that derive biosynthetically from ubiquitous porphyrins. The investigations of phytochromes reveal that precise interactions between the protein moiety and its bilin chromophore are essential for the proper functioning of this photoreceptor; accordingly, synthetic manipulation of the parts is an important method for studying the whole. Although variations in the protein structure are readily accomplished by routine mutagenesis protocols, the generation of structurally modified bilins is a laborious, multistep process. Recent improvement in the synthesis of open-chain tetrapyrroles now permits the generation of novel, structurally modified (and even selectively isotope-labeled) chromophores. Furthermore, by using the capability of recombinant apo-phytochrome to bind the chromophore autocatalytically, researchers can now generate novel chromoproteins with modified functions. In the protein-bound state, the phytochrome chromophore is photoisomerized at one double bond, in the bridge between the last two of the four pyrrole rings (the C and D rings), generating the thermally stable, physiologically active P(fr) form. This conversion--photoisomerization from the form absorbing red light (P(r)) to the form absorbing far-red light (P(fr))--covers 12 orders of magnitude, from subpicoseconds to seconds. Such spectroscopic and kinetic studies yield a wealth of time-resolved spectral data, even more so, if proteins with changed sequence or chromophore structure are utilized. In particular, bilins with a changed substitution pattern at the photoisomerizing ring D have shed light on the chromophore-protein interactions during the photoisomerization. The mechanisms generating and stabilizing the light-induced P(fr) form of phytochromes are now seen in greater detail. On the other hand, the use of bilins with selective incorporation of stable isotopes identify light-induced conformational motions when studied by vibrational (FTIR and Raman) and NMR spectroscopy. In this Account, we present spectroscopic investigations that provide structural details in these biological photoreceptors with great precision and document the dynamics elicited by light excitation. This approach yields important information that complements the data deduced from crystal structure.

Chromophore structure of cyanobacterial phytochrome Cph1 in the Pr state: reconciling structural and spectroscopic data by QM/MM calculations.

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His(260) in the chromophore-binding pocket such that either the delta-nitrogen (M-HSD) or the epsilon-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His(260) with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.

Interactions between chromophore and protein in phytochrome identified by novel oxa-, thia- and carba-chromophores.

Six new bilin chromophores of the plant photoreceptor phytochrome have been synthesized, carrying at the photoisomerizing ring D an oxygen or a sulfur atom or a methylene group instead of the pyrrole nitrogen atom. These furanone-, thiophenone- or cyclopentenone-containing compounds bound covalently to the recombinant apophytochrome phyA of Avena sativa. The novel chromoproteins showed hypsochromically shifted absorption spectra with respect to native phytochrome and a strongly diminished photochemical activity, but a three- to four-fold higher fluorescence quantum yield. These results demonstrate that, on the one hand, also ring D-modified chromophores can be forced into a partially extended structure, required for incorporation into the apoprotein binding pocket and covalent binding. On the other hand, the modifications introduced into ring D of the chromophores strongly impede the formation of stable far red-absorbing forms of plant photoreceptor phytochrome (P(fr)-form) of the chromoproteins, highlighting especially the role of the pyrrole nitrogen atom and hydrogen bonding for the precise interactions between that part of the chromophore and the protein for the P(fr)-formation.