An Alternative Mechanism of Bioluminescence Color Determination in Firefly Luciferase

Abstract

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

“…Recently, however, Branchini and coworkers suggested that luciferase modulates emission color by controlling the resonancebased charge delocalization of the anionic keto form of excited oxyluciferin (28). Theoretical studies indicated that the degree of polarization of oxyluciferin phenolate/ keto-enolate groups, under influence of the active site residues, could be determinant of bioluminescence colors (27).…”

“…Considering that the benzothiazolyl moietyis tightly buried and stabilized in the binding site, through hydrophobic forces and phenolate interaction with R215, it is possible that at least during the emitting step, the thiazolinic moiety could be exposed to a more polar environment in the case of PxRE luciferase and to a more hydrophobic environment in the case of PxGR luciferase. According to the recently proposed theory of polarization of phenolate and keto/enolate groups (27,28), the substitution of R215 would be expected to affect the emission spectrum by increasing the polarization of excited oxyluciferin, if the keto/enolate moiety is not polarized. This is supported by the experimental observation that the substitution of R215 affects the bioluminescence spectrum only in the green emitting railroadworm and firefly luciferases, but not in the red emitting PxRE luciferase.…”

“…Three mechanisms have been proposed to explain bioluminescence colour modulation by beetle luciferases: (I) non-specific solvent and orientation polarizability effects (15,16); (II) specific active-site interactions with the emitter (25); and (III) the effect of the conformation of the active site on the rotational properties of the oxyluciferin thiazine rings (26). Recent theoretical and experimental studies suggested that the polarization of the phenolate and enolate groups of excited oxyluciferin under the influence of active site residues determine of the emission spectra (27,28). However, the contribution of each mechanism to the determination of the bioluminescence colours produced by different beetle luciferases is unclear.…”

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

“…Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.The most red-shifted luciferin analogues to date [16] are based upon amino derivatives (Figure 1 b), for example cyclic aminoluciferin (3 a: l max = 599 nm; 3 b: l max = 607 nm), [17] seleno-d-aminoluciferin (4: l max = 600 nm), [18] and a rationally designed 4-(dimethylamino)phenyl derivative conjugated to a thiazoline group (5: l max = 675 nm). [19] In particular cyclic aminoluciferin derivative 3 a has been shown to give improved bioluminescence imaging compared to luciferin (LH 2 ; 1) at dilute concentrations where the intracellular concentration of the luciferin or analogue is limiting.…”

“…Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.…”