Bacterial luciferase requires one reduced flavin for light emission.

Abstract

Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A twoflavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. (4) indicated that the bioluminescent reaction had a second order dependence on FMNH2 concentration, again suggesting the involvement of two flavins.Some years later, when the cad heterodimeric structure of luciferase was elucidated (5), the possibility that two reduced flavins might be required for activity could be envisioned in more specific terms. However, binding site determinations by a kinetic method showed that luciferase possesses only a single FMNH2 binding site per dimer (6). Furthermore, chemical modification studies (7,8) and mutant analyses (9) indicated that only the a subunit participates in the catalytic steps. A reaction mechanism involving the oxidation of a single FMNH2 and the concomitant oxidation of aldehyde was thus proposed (6, 10) and has been supported by a variety of experimental results since then (1, 11), including the demonstration that aldehyde is indeed oxidized to acid (12-15). However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 based on an absorptivity of 12,500 M-1 cm-1 at 445 nm (23). 2,2-Bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (BisTris) and dithiothreitol were obtained from Sigma, decanal from Aldrich, bovine serum albumin from Pentex, dithionite (sodium hydrosulfite) from Mallinckrodt. Absorbance values were read with a Cary 15 spectrophotometer, circular dichroism spectra with a Jasco J-20 spectropolarimeter. Fig. 1 gives circular dichroism (CD) spectra for free FMNH2, free luciferase, and an equimolar mixture of FMNH2 and luciferase, all three in the presence of dithionite. The relatively large signal at 370 nm for the mixture is indicative of a complex between reduced flavin and luciferase. The stoichiometry of this complex was examined by the method of continuous variation of mole fraction according to job (24). When equimolar solutions of FMNH2 and luciferase are mixed in different proportions, the CD signal at 370 nm varies as a function of the relative mole fraction of the reactants (Fig. 2). Maximum signal develops for a mole fraction of flavin (and enzyme) near 0.5, indicating that luciferase has one binding site for FMNH2. Although these data are not ideal for the calculation of a dissociation constant Kd for the E:FMNH2 complex, they are consistent with the published value of 0.8 ,M (6). RESULTS AND DISCUSSION

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

“…Flavoenzymes are a large and structurally diverse class of enzymes, catalysing a wide range of biochemical reactions involved in many processes, including oxidation and dehydrogenation of metabolites, light emission1, energy production2, DNA repair3 and apoptosis4. The diversity of these functions is due to the versatility of the flavin cofactor which may undergo the redox cycle through one or two electron transfers5.…”

The 1.5-Å Resolution Crystal Structure of Bacterial Luciferase in Low Salt Conditions

“…The two polypeptides, encoded on adjacent genes, luxA and luxB in the lux operon, display sequence homology and appear to have arisen by gene duplication (4). There is a single active center in the luciferase heterodimer that resides on the ␣ subunit (5) and binds one reduced flavin molecule (6,7). The role of the ␤ subunit is not clear at this time but is essential for a high quantum yield reaction (8).…”

Bioluminescence and Chemiluminescence

“…The reaction may be viewed as a monooxygenase [97] with the following overall stoichiometry and a chemiluminescent quantum yield of about 0.2 [16,114].…”