Luciferase

The luciferases of fireflies – of which there are over 2000 species – and of the other Elateroidea (click beetles and relatives in general) are diverse enough to be useful in molecular phylogeny.[5] In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. One well-studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.[6]

Also well studied is the sea pansy, Renilla reniformis. In this organism, the luciferase (Renilla-luciferin 2-monooxygenase) is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:[7]

• coelenterazine + O₂ → coelenteramide + CO₂ + photon of light

Newer luciferases have recently been identified that, unlike other luciferases, are naturally secreted molecules. One such example is the Metridia coelenterazine-dependent luciferase (MetLuc, A0A1L6CBM1) that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.[8]

Bacterial bioluminescence is seen in Photobacterium species, Vibrio fischeri, Vibrio haweyi, and Vibrio harveyi. Light emission in some bioluminescent bacteria utilizes 'antenna' such as lumazine protein to accept the energy from the primary excited state on the luciferase, resulting in an excited lulnazine chromophore which emits light that is of a shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with flavin mononucleotide (FMN) as the chromophore and emits light that is red-shifted relative to that from luciferase.[9]

Dinoflagellate luciferase is a multi-domain eukaryote protein, consisting of an N-terminal domain, and three catalytic domains, each of which preceded by a helical bundle domain. The structure of the dinoflagellate luciferase catalytic domain has been solved.[10] The core part of the domain is a 10 stranded beta barrel that is structurally similar to lipocalins and FABP.[10] The N-terminal domain is conserved between dinoflagellate luciferase and luciferin binding proteins (LBPs). It has been suggested that this region may mediate an interaction between LBP and luciferase or their association with the vacuolar membrane.[11] The helical bundle domain has a three helix bundle structure that holds four important histidines that are thought to play a role in the pH regulation of the enzyme.[10] There is a large pocket in the β-barrel of the dinoflagellate luciferase at pH 8 to accommodate the tetrapyrrole substrate but there is no opening to allow the substrate to enter. Therefore, a significant conformational change must occur to provide access and space for a ligand in the active site and the source for this change is through the four N-terminal histidine residues.[10] At pH 8, it can be seen that the unprotonated histidine residues are involved in a network of hydrogen bonds at the interface of the helices in the bundle that block substrate access to the active site and disruption of this interaction by protonation (at pH 6.3) or by replacement of the histidine residues by alanine causes a large molecular motion of the bundle, separating the helices by 11Å and opening the catalytic site.[10] Logically, the histidine residues cannot be replaced by alanine in nature but this experimental replacement further confirms that the larger histidine residues block the active site. Additionally, three Gly-Gly sequences, one in the N-terminal helix and two in the helix-loop-helix motif, could serve as hinges about which the chains rotate in order to further open the pathway to the catalytic site and enlarge the active site.[10]

A dinoflagellate luciferase is capable of emitting light due to its interaction with its substrate (luciferin) and the luciferin-binding protein (LBP) in the scintillon organelle found in dinoflagellates.[10] The luciferase acts in accordance with luciferin and LBP in order to emit light but each component functions at a different pH. Luciferase and its domains are not active at pH 8 but they are extremely active at the optimum pH of 6.3 whereas LBP binds luciferin at pH 8 and releases it at pH 6.3.[10] Consequently, luciferin is only released to react with an active luciferase when the scintillon is acidified to pH 6.3. Therefore, in order to lower the pH, voltage-gated channels in the scintillon membrane are opened to allow the entry of protons from a vacuole possessing an action potential produced from a mechanical stimulation.[10] Hence, it can be seen that the action potential in the vacuolar membrane leads to acidification and this in turn allows the luciferin to be released to react with luciferase in the scintillon, producing a flash of blue light.

The reaction catalyzed by bacterial luciferase is also an oxidative process:

• FMNH₂ + O₂ + RCHO → FMN + RCOOH + H₂O + light

In the reaction, molecular oxygen oxidizes flavin mononucleotide and a long-chain aliphatic aldehyde to an aliphatic carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.[12]

Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%[13] to 90%[14] efficient. In comparison, the incandescent light bulb only converts about 10% of its energy into light[15] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.[14]