Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations

Abstract

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.

The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

“…The same conclusion can be drawn for all P2PG or iRFP variants. Small frequency variations observed for the N-H ip indicate minor changes of the hydrogen bond interactions of the ring B and C N-H groups25.…”

“…For a detailed vibrational assignment we therefore refer to previous analyses2526. In this work we focus on the identification of selected modes that correlate with specific structural parameters of the tetrapyrrole.…”

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

“…The average Raman spectrum of the protein-bound BV chromophore was calculated by summing up the Raman spectra computed at a QM/MM level for each of the snapshots following published protocols (23,(47)(48)(49). It has been demonstrated for other phytochromes that calculated (off-resonance) Raman intensities are a good approximation for Raman spectra measured under rigorous resonance and pre-resonance excitation (50,51).…”

“…Starting with the crystallographic structure of the Pfr state of the bathy phytochrome from PaBphP (14), molecular dynamics (MD) simulations combined with calculations using molecular mechanical-quantum mechanical (QM/ MM) hybrid methods were carried out to achieve a structural refinement for the chromophore binding pocket, guided by a comparison of the calculated and the experimental resonance Raman (RR) spectra of the tetrapyrrole cofactor (23). Specifically, we asked whether the results of the present approach were consistent with the original assignment of the bilin structure to a ZZEssa configuration (14), a plausible assignment albeit not unambiguously backed by the rather disperse electron density data, which in principle would also be compatible with a ZZZssa geometry from protein crystallography (14).…”

NMR Spectroscopic Investigation of Mobility and Hydrogen Bonding of the Chromophore in the Binding Pocket of Phytochrome Proteins

“…[20][21][22] Information about chromophore-protein interactions at atomic resolution is essential for a detailed understanding of the photoconversion mechanism. While spectroscopic techniques provided insights into the molecular structure and led to the creation of structural models, [23][24][25][26][27][28][29][30][31] several 3D structures from X-ray diffraction and NMR studies are now available. [32][33][34][35][36][37] They confirm that the major structural change taking place during photoconversion is the isomerization of the double bond between the rings C and D (Figure 1), thus converting the conformation of the chromophore from ZZZssa to ZZEssa.…”