Theoretical Study of Fluorescence Spectra Utilizing the pK_a Values of Acids in Their Excited States

Abstract

Assignment of the fluorescence spectrum of firefly luciferin in aqueous solutions was achieved by utilizing not only emission energies but also theoretical absorption spectra and relative concentrations as estimated by pKa values. Calculated Gibbs free energies were utilized to estimate pKa values. These pKa values were then corrected by employing the experimental results. It was previously thought that the main peak near 550 nm observed in the experimental fluorescence spectra at all pH values corresponds to emission from the first excited state of the luciferin dianion [Ando et al. (2010) Jpn. J. Appl. Phys. 49, 117002-117008]. However, we found that the peak near 550 nm at low pH corresponds to emission from the first excited state of the phenolate monoanion of luciferin. Furthermore, we found that the causes of the red fluorescence at pH 1-2 are not only the emission from phenol monoanion but also the emission from the protonated species at nitrogen atom in the thiazoline ring of dianion.

Analysis of Photoexcitation Energy Dependence in the Photoluminescence of Firefly Luciferin

“…In this study, we assume that only a single protonation or deprotonation event occurs in S 1 . The same assumption was made in our previous paper and successful results were obtained.…”

“…Our previous paper reported the change in Gibbs free energy (Δ G) for eq. (1) calculated using the results of vibrational analysis for luciferin and for its conjugate acids and bases in S 1 . The relationship between Δ G and concentration ([A − ] and [AH]) is written aswhere R denotes the universal gas constant and T denotes the temperature.…”

“…Many experimental and theoretical studies for the absorption and fluorescence spectra of luciferin have been reported (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Biltler and McElroy first reported the spectroscopic study upon purification and crystallization of luciferin molecules (10) and this was followed by experimental studies of photochemical properties of luciferin molecule as a substrate in bioluminescence as well as a reactant in chemiluminescence (11,12).…”

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

“…However, although we have reason to believe from the preceding benchmark calculations that these values, which are included in Table S9 of the Supporting Information, offer a reliable description of the excited‐state reactivity of OxyLH 2 , we will instead base our analysis on a set of excited‐state p K values obtained in a different way (importantly, the resulting data and the data in Supporting Information Table S9 sustain the same exact conclusion on the identity of the preferred OxyLH 2 species). Specifically, as alluded to in the previous section and as argued also by other authors, it is to some extent possible to cancel inevitable computational errors in p K E BH (S 1 ) and p K a BH (S 1 ) by rather considering the p K BH (S 1 ) values, henceforth denoted p K BH,corr (S 1 ), obtained by adding calculated Δp K BH (S 1 ) values to experimental ground‐state p K values [p K exp (S 0 )] …”

“…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).…”