Action spectroscopy of isomer-selected luciferin anions

Abstract

Luciferin molecules are common luminophores found throughout the biological kingdoms. Here, electrospray ionization and tandem ion mobility spectrometry coupled with laser spectroscopy are used to demonstrate that D-luciferin and oxyluciferin deprotonated anions can be produced in two isomeric forms, which can be separated by virtue of their different collision cross sections with a buffer gas. The two isomers possess distinguishable but partially overlapping photodepletion action spectra over the visible range, implying distinct intrinsic absorption profiles. The site of deprotonation and tautomeric forms of the electrosprayed isomers are assigned through comparisons between experimental and calculated collision cross sections and electronic excitation energies. The study clearly shows that electrospray ionization of biochromophore molecules can generate multiple isomeric forms with distinct electronic spectra.

Intrinsic fluorescence from firefly oxyluciferin monoanions isolated in vacuo

“…According to ion-mobility spectroscopy experiments performed by Bieske and co-workers, 22 the absorption by the enol form is minimal when the excitation wavelength is higher than 550 nm while the keto form absorbs maximally around 540 nm. Hence with 561 nm light for photoexcitation (chosen based on available notch filters), we basically only excite the keto form, and indeed the fluorescence spectra corresponding to the lower-energy band are very similar to that of dm-OL − (Fig.…”

“…OL − monoanions are formed predominantly in the keto and enol forms by electrospray ionization according to previous work. 22,23 The ratio between the two tautomers is determined by the solvent: the keto form dominates with methanol (MeOH) as solvent, and its yield increases even more relative to the enol form when acetonitrile (MeCN) is used. Indeed, electrospray ionization is known to affect the ratio of isomers of small organic molecules, see e.g.…”

“…3(d)). A comparison with the spectrum of dm-OL − and the fact that the enol form absorbs to the blue of the keto form 22 indicate that the two bands are due to the enol form and the keto form, respectively. In accordance with this interpretation, the 597 nm band increases in importance relative to the 563 nm band with MeCN as the solvent for electrospray (Fig.…”

“…The ion source is similar to that used for our previous action-spectroscopy experiments, and there it was concluded that the keto form is the major species. 17,22 Also pretrapping of ions in an octopole was done in the earlier experiment. An explanation could be phototautomerization, but this channel can be excluded based on ion-mobility spectrometry.…”

“…These experiments clearly demonstrated that the transition energy strongly depends on hydrogen-bond and electrostatic interactions within the luciferase pocket where oxyluciferin resides. Recently, Bieske and co-workers 22 used ion mobility spectrometry to separate the keto and enol forms and measured action spectra of each tautomer. They found that the ketone absorbs maximally at ≈540 nm and the enol at ≈475 nm, but that the spectra in both cases are broad and somewhat overlap.…”