**2.1 ATP-Bioluminescence principle**

Light-producing living organisms are widespread in nature and from diverse origins. The process of light emission from organisms is called bioluminescence and represents a chemical conversion of energy into light. Since the work of William D McElroy showing that ATP is a limiting and key factor of the bioluminescent reaction, research has lead to a better understanding of how light is produced by fireflies (McElroy, 1947; McElroy, 1951; McElroy et al., 1953). The bioluminescence mechanism involving Luciferase enzyme is a multistep process which mainly requires Luciferin substrat, Oxygen (O2), Magnesium cation (Mg++) and ATP (DeLuca & McElroy, 1974; McElroy et al., 1953; Seliger, 1989). ATPbioluminescence using luciferine/luciferase relies on luciferine oxidation by the luciferase and the integrated light intensity is directly proportional to ATP contents. Luciferase converts in presence of ATP and Magnesium firefly D-luciferin into the corresponding enzyme-bound luciferil adenylate. The luciferil adenylate complex is then the substrate of the subsequent oxidative reaction leading to oxyluciferin. The light emission is a consequence of a rapid loss of energy of the oxyluciferine molecule from an excited state to a stable one. This reaction induces the emission of photons with a efficient quantum yield of about 90% (Seliger, 1989; Wilson & Hasting, 1998) (Fig1).

1/ D-luciferin + luciferase + ATP Mg Luciferil adenylate complex +PPi

2/ Luciferil adenylate complex *<sup>O</sup>* 2 Oxyluciferin + AMP+ CO2 + light

Fig. 1. Chemical reactions of the ATP-bioluminescence based on luciferin/luciferase system (PPi:inorganic pyrophosphate, CO2: Carbon Dioxide). Photons of yellow-green light (550 to 570 nm) are emitted.

#### **2.2 Luciferase protein**

Luciferase is a common term used to describe enzymes able to catalyze light emission. Luciferase belongs to the adelynate-forming protein family and is an oxygen-4 oxidoreductase gathering decarboxylation and ATP-hydrolysing main activities. Structural studies have shown that Photinus pyralis Luciferase protein is folded into 2 domains: a large N-terminal body and a small C-terminal domain linked by a flexible peptide creating a wide cleft (Conti et al., 1996). Amino acids critical for bioluminescence phenomenon belong mainly to the N-terminal domain (Branchini et al., 2000; Thompson et al., 1997; Zako et al., 2003). This implies that luciferine-binding site is mediated by conformational change to bring the 2 domains closer. This conformational change is consistent with the study of Nakatsu et al (2006) showing that luciferase from luciola cruciata exists in an "open form" and in a "closed form", the later form creates an hydrophobic pocket around the active site and is responsible of light emission. Two kinds of colored light emission are described for luciferine/luciferase reaction. The typical high energy yellow-green light emission with a peak at 562 nm at pH 7.5 and red light emission with a peak at 620nm when the pH decreases to 5 (Seliger et al., 1964; Seliger & McElroy, 1964). This surprising phenomenon where Luciferase is able to emit light of different colors is not clearly understood but the isolation of colored luciferase variants shows that single amino acid substitution in N-terminal domain affects bioluminescence color by modulating slightly the polarity of the active site environment (Hosseinkhani, 2011; Shapiro et al., 2005). This interesting feature opens the way to wide applications in biotechnology (Branchini et al., 2005).
