On the Influence of Water on the Electronic Structure of Firefly Oxyluciferin Anions from Absorption Spectroscopy of Bare and Monohydrated Ions in Vacuo

Abstract

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

Unraveling the Intrinsic Color of Chlorophyll

“…[21] The range-separated CAM-B3LYP functional [22,23] was chosen as this has previously been shown [24] to provide results similar to those obtained with the computationally demanding equation of motion coupled-cluster level of theory. [21] The range-separated CAM-B3LYP functional [22,23] was chosen as this has previously been shown [24] to provide results similar to those obtained with the computationally demanding equation of motion coupled-cluster level of theory.…”

Distinguishing between keto-enol and acid-base forms of firefly oxyluciferin through calculation of excited-state equilibrium constants

“…One bioluminescent reaction system with a particularly high quantum yield is that of fireflies, which has been the topic of many recent experimental and theoretical studies . However, despite that much progress has been made in the elucidation of the mechanisms underlying the light emission of fireflies, many details of the luciferase‐catalyzed formation of the chemiexcited (S 1 , first excited singlet state) oxyluciferin emitter (OxyLH 2 ) from d ‐luciferin (LH 2 , a ground‐state species), are yet to be resolved. As shown in Figure and described in detail elsewhere, this conversion is initiated by adenylation of LH 2 with ATP‐Mg 2+ , which forms d ‐luciferyl‐adenylate (LH 2 ‐AMP).…”

Photoinduced dissociation mass spectroscopy of firefly oxyluciferin anions

“…An environment can provide an efficient way of protection by energy dissipation [10]. The dissociation time of the isolated photoexcited ions in vacuo is about 100 s based on previous storage-ring experiments [11], which results in a dissociation yield of 10 -6 -10 -8 assuming a time constant for vibrational cooling between 1 ps and 100 ps. Each flash from a firefly contains about 10 13 photons [12], and the same order of magnitude of hot ions is therefore produced (59 % yield versus 41 %).…”

“…A wavelength of 550 nm was used as we have earlier found that this is the maximum of the absorption band [11]. For each of the identified fragments, we calculated the reaction energy associated with the dissociation channel to see if the reaction would be possible or not for A c c e p t e d M a n u s c r i p t 4 vibrationally excited (hot) oxyluciferin ions within the luciferase enzyme.…”

“…For each of the identified fragments, we calculated the reaction energy associated with the dissociation channel to see if the reaction would be possible or not for A c c e p t e d M a n u s c r i p t 4 vibrationally excited (hot) oxyluciferin ions within the luciferase enzyme. The lowest-energy structure of oxyluciferin is the keto form [11] but the enol form ( Fig. 1) may also be present in the ion beam.…”