Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar environment weakens such interaction promoting red shifts. In pH-sensitive luciferases, a pH-mediated switch from a closed hydrophobic conformation to a more open polar conformation promotes the typical red shift.

Spectroscopic properties of amino-analogs of luciferin and oxyluciferin were investigated to confirm the color modulation mechanism of firefly (beetle) bioluminescence. Fluorescence solvatochromic character of aminooxyluciferin analogs indicates that the bioluminescence of aminoluciferin is useful for evaluating the polarity of a luciferase active site.

How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6′-substituted aminoluciferin analogues (6′-morpholino- and 6′-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the K M and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6′-morpholino- (634 nm) and 6′-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6′-aminoluciferins provide potential far-red combinations for bioimaging applications.

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