Spectroscopic and Photophysical Investigation of Model Dipyrroles Common to Bilins: Exploring Natural Design for Steering Torsion to Divergent Functions

Abstract

Biliproteins are a unique class of photosynthetic proteins in their diverse, and at times, divergent biophysical function. The two contexts of photosynthetic light harvesting and photoreception demonstrate characteristically opposite criteria for success, with light harvesting demanding structurally-rigid chromophores which minimize excitation quenching, and photoreception requiring structural flexibility to enable conformational isomerization. The functional plasticity borne out in these two biological contexts is a consequence of the structural plasticity of the pigments utilized by biliproteins―linear tetrapyrroles, or bilins. In this work, the intrinsic flexibility of the bilin framework is investigated in a bottom-up fashion by reducing the active nuclear degrees of freedom through model dipyrrole subunits of the bilin core and terminus free of external protein interactions. Steady-state spectroscopy was carried out on the dipyrrole (DPY) and dipyrrinone (DPN) subunits free in solution to characterize their intrinsic spectroscopic properties including absorption strengths and nonradiative activity. Transient absorption (TA) spectroscopy was utilized to determine the mechanism and kinetics of nonradiative decay of the dipyrrole subunits, revealing dynamics dominated by rapid internal conversion with some Z→E isomerization observable in DPY. Computational analysis of the ground state conformational landscapes indicates enhanced complexity in the asymmetric terminal subunit, and the prediction was confirmed by heterogeneity of species and kinetics observed in TA. Taken together, the large oscillator strengths (f ∼ 0.6) of the dipyrrolic derivatives and chemically-efficient spectral tunability seen through the ∼100 nm difference in absorption spectra, validate Nature's "selection" of multi-pyrrole pigments for light capture applications. However, the rapid deactivation of the excited state via their natural torsional activity when free in solution would limit their effective biological function. Comparison with phytochrome and phycocyanin 645 crystal structures reveals binding motifs within the in vivo bilin environment that help to facilitate or inhibit specific inter-pyrrole twisting vital for protein operation.

Photoisomerization of phytochrome chromophore models: An XMS-CASPT2 study

“…Zietz and Blomgren 28 used a dipyrrinone (DPN) model of bilirubin where all the alkyl groups were replaced by hydrogen atoms. Dean and coworkers 27 reported a combined spectroscopic and computational study of two truncated models: DPN and dipyrrole (DPY).…”

“…Klan and coworkers 29,37 measured transient absorption spectra of a fragment of bilirubin containing the C-and D-rings with the functional groups as found in bilirubin. In all these studies discussed above, only the DPY model of Dean and coworkers 27 is positively charged, while all the other chromophores are neutral.…”

“…19 Therefore, in previous studies a compromise was chosen to either employ approximate electronic structure methods, such as a semi-empirical method, [20][21][22] or to truncate the chromophore. [23][24][25][26][27][28] In the latter approach, typically, the propionic acid groups 23 are replaced by hydrogen atoms because they are not part of the conjugated π-system. Other strategies to reduce the model size include removal of methyl groups 24,25 or even truncation of the pyrrole rings.…”

“…Other strategies to reduce the model size include removal of methyl groups 24,25 or even truncation of the pyrrole rings. 26,27 Zietz and Blomgren 28 used the CASSCF method with varying active spaces to locate CIs on a truncated model comprising of two pyrrole rings in the gas phase. They found that the calculated CIs were 12 kcal•mol -1 lower in energy than the FC point and can be reached by a concerted twist of the 𝐶 14 − 𝐶 15 and 𝐶 15 = 𝐶 16 bonds.…”

“…Recently, truncated tetrapyrrole models have been also studied experimentally. 27,29,[36][37][38] Ultrafast spectroscopy experiments of model chromophores in solution have been reported. These experiments have found an ultrafast relaxation of the chromophore where the isomerization takes place on a picosecond timescale.…”