Red-light absorption and fluorescence of phytochrome chromophores: A comparative theoretical study

Abstract

Currently, much experimental effort is being invested in the engineering of phytochromes, a large superfamily of photoreceptor proteins, into fluorescent proteins suitable for bioimaging in the near-infrared regime. In this work, we gain insight into the potential of computational methods to contribute to this development by investigating how well representative quantum chemical methods reproduce recently recorded red-light absorption and emission maxima of synthetic derivatives of the bilin chromophores of phytochromes. Focusing on the performance of time-dependent density functional theory but using also the ab initio CIS(D), CC2 and CASPT2 methods, we explore how various methodological considerations influence computed spectra and find, somewhat surprisingly, that density functionals lacking exact exchange reproduce the experimental measurements with smaller errors than functionals that include exact exchange. Thus, for the important class of chromophores that bilins constitute, the widely established trend that hybrid functionals give more accurate excitation energies than pure functionals does not apply.

How Method-Dependent Are Calculated Differences between Vertical, Adiabatic, and 0–0 Excitation Energies?

“…Among the hybrid functionals, those that include a small fraction of exact exchange display negative or slightly negative MSEs of −0.10 (B3LYP) and −0.02 eV (PBE0), whereas those that include a large fraction show positive MSEs of 0.07−0.13 eV (M06-2X, M06-HF, CAM-B3LYP, and ωB97X-D). These trends are consistent with the observation in a previous TD-DFT benchmark concerned entirely with ΔE ve energies that (albeit with some notable exceptions 90 ) pure functionals like BP86 typically redshift, and the inclusion of successively larger fractions of exact exchange in hybrid functionals typically blue-shift, the excitation energies of organic molecules. 12 CIS and CC2, in turn, show positive MSEs for the organic systems.…”

QM/MM Benchmarking of Cyanobacteriochrome Slr1393g3 Absorption Spectra

“…Furthermore, we stress that caution should be exercised, when TD-DFT is employed: A tuning similar to the one from the aforementioned methods is only obtained with a range-separated hybrid functional, but then excitation energies are overestimated. On the other hand, BLYP as a prototypical GGA functional leads to a negligible tuning, but it is the best functional to calculate the absorption spectrum for the P r form in agreement with previous benchmark studies [44,51]. Furthermore the root mean square electron hole separation, which can be interpreted as exciton size [92], for the first excited state S 1 obtained as an average over 10 snapshots for P r increases from CAM-B3LYP (5.06 Å) to B3LYP (5.90 Å) to BLYP (6.16 Å), whereas the corresponding result for RI-ADC(2) is 5.11 Å, see Table S15.…”

“…In particular, Durbeej et al investigated phytochromobilin and reported among others that not including the thioether linkage, the propionate side chains and the methyl groups has only a small influence on excitation energies and oscillator strengths [42,43]. This was later extended by a benchmark study of locked bilin chromophores, where it was found that TD-DFT with pure generalized gradient approximation (GGA) functionals is a particularly viable choice of methodology for calculating absorption and emission maxima of bilin chromophores [44]. Furthermore, Matute et al employed TD-DFT to predict the chromophore conformations, when they are bound to the proteins [21,45].…”

A Hydrogen Bond Between Linear Tetrapyrrole and Conserved Aspartate Causes the Far-Red Shifted Absorption of Phytochrome Photoreceptors

“…The experimental Qband shifts were also reproduced by the TD-CAM-B3LYP 21 calculations ( Figure 2b) although the computed energies are systematically higher than the experimental ones. The overestimation of the energies with a TD-DFT method is consistent with previous reports [22][23] . The Soret-band maximum (350-420 nm) cannot be reliably predicted by the TD-CAM-B3LYP calculations, indicating contributions from double-and higher-order excitations.…”