The chemical basis of the blue-black to pink-orange color change on cooking of lobster, due to thermal denaturation of an astaxanthin-protein complex, alpha-crustacyanin, in the lobster carapace, has so far been elusive. Here, we investigate the relaxation of the astaxanthin pigment from its bound enolate form to its neutral hydroxyketone form, as origin of the spectral shift, by analyzing the response of UV-vis spectra of a water-soluble 3-hydroxy-4-oxo-beta-ionone model of astaxanthin to increases in pH, and by performing extensive quantum chemical calculations over a wide range of chemical conditions. The enolization of astaxanthin is consistent with the X-ray diffraction data of beta-crustacyanin (PDB code: 1GKA) whose crystals possess the distinct blue color. We find that enolate formation is possible within the protein environment and associated with a large bathochromic shift, thus offering a cogent explanation for the blue-black color and the response to thermal denaturation and revealing the chemistry of astaxanthin upon complex formation.

Although recent years have seen much progress in the elucidation of the mechanisms underlying the bioluminescence of fireflies, there is to date no consensus on the precise contributions to the light emission from the different possible forms of the chemiexcited oxyluciferin (OxyLH(2)) cofactor. Here, this problem is investigated by the calculation of excited-state equilibrium constants in aqueous solution for keto-enol and acid-base reactions connecting six neutral, monoanionic and dianionic forms of OxyLH(2). Particularly, rather than relying on the standard Forster equation and the associated assumption that entropic effects are negligible, these equilibrium constants are for the first time calculated in terms of excited-state free energies of a Born-Haber cycle. Performing quantum chemical calculations with density functional theory methods and using a hybrid cluster-continuum approach to describe solvent effects, a suitable protocol for the modeling is first defined from benchmark calculations on phenol. Applying this protocol to the various OxyLH(2) species and verifying that available experimental data (absorption shifts and ground-state equilibrium constants) are accurately reproduced, it is then found that the phenolate-keto-OxyLH(-) monoanion is intrinsically the preferred form of OxyLH(2) in the excited state, which suggests a potential key role for this species in the bioluminescence of fireflies.

Electronic transitions from one excited state to another excited state of different spin symmetry play important roles in many biochemical reactions. Although recent years have seen much progress in the elucidation of nonradiative (intersystem crossing) relaxation mechanisms for such transitions, there is presently a scarcity of data available to assess whether also radiative (phosphorescence) mechanisms are relevant for these processes. Here, we demonstrate that the well-established ability of quantum chemical methods to describe intersystem crossing events between excited states, can be supplemented by the ability to also describe inter-excited state phosphorescence. Specifically, performing four-component relativistic time-dependent density functional theory calculations, we obtain rate constants for the radiative transitions from the absorbing ¹(πHπL*) singlet state of lumiflavin to the ³(πHπL*), ³(nN2πL*) and ³(πH–1πL*) triplet states, and subsequently compare these results with rate constants calculated for the corresponding nonradiative transitions. Thereby, it is found that the radiative rate constants for these particular transitions are typically two to five orders of magnitude smaller than the nonradiative ones.

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.

Phytochromes constitute one of the six well-characterized families of photosensory proteins in Nature. From the viewpoint of computational modeling, however, phytochromes have been the subject of much fewer studies than most other families of photosensory proteins, which is likely a consequence of relevant high-resolution structural data becoming available only in recent years. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate UV-vis absorption spectra of Deinococcus radiodurans bacteriophytochrome. We investigate how the choice of QM/MM methodology affects the resulting spectra and demonstrate that QM/MM methods can reproduce the experimental absorption maxima of both the Q and Soret bands with an accuracy of about 0.15 eV. Furthermore, we assess how the protein environment influences the intrinsic absorption of the bilin chromophore, with particular focus on the Q band underlying the primary photochemistry of phytochromes.

Phytochromes constitute a superfamily of photoreceptor proteins that exist in two forms that absorb red (Pr) and far-red (Pfr) light. The conversion of Pr into Pfr (the biologically active form) is triggered by a Z → E photoisomerization of the bilin chromophore at the C15-C16 bond of the methine bridge between pyrrole rings C and D. Here, we present hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on a highresolution Pr crystal structure of Deinococcus radiodurans bacteriophytochrome to investigate the competition between all possible photoisomerizations at the three different (AB, BC and CD) methine bridges. The results demonstrate that steric interactions with the protein are a key discriminator between the different reaction channels. In particular, it is found that such interactions prevent photoisomerization at any other site than the C15-C16 bond. The tendency of phytochromes to always isomerize at this very bond would thus be explained by steric effects.