Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

Abstract

ABSTRACT:The bioluminescent reaction of luciferase isolated from Vibrio harveyi, strain M17, was initiated by mixing the luciferase-bound flavin 4a-hydroperoxide intermediate, purified in advance, with a long-chain aldehyde (dodecanal or octanal) at -4 O C . Measurements of absorbance changes from 300 to 600 nm during the course of the reaction revealed the existence of three sets of isosbestic points and three kinetic phases, the second of which parallels kinetically the decay of bioluminescence, measured concurrently. The absorbance changes in this second step and the decay of light emission exhibited similar deuterium isotope effects; this is postulated to be the step giving rise to the excited state and the enzyme-bound flavin 4a-hydroxide. The first step of the reaction, however, did not show an isotope effect; the intermediate thereby formed, observed here for the first time, is postulated to correspond to the luciferase-bound flavin 4a-peroxyhemiacetal.Of the many light-emitting organisms, only the bacteria produce a luciferase that utilizes a flavin as a substrate and the emitter (Hastings, 1983). Bacterial luciferase (EC 1.14.14.3) is a monooxygenase which gives rise to light emission upon reaction of enzyme-bound FMNH2' with molecular oxygen and a long-chain aldehyde. The products are oxidized FMN, the corresponding fatty acid, and water (Hastings et al., 1985;Baldwin & Ziegler, 1991). Thepostulatedpathway and principal intermediates (all luciferase-bound) are shown in Scheme 1.A key intermediate (11), which is formed by reaction of FMNH2 with oxygen and occurs prior to reaction with aldehyde, is the flavin 4a-hydroperoxide (Hastings et al., 1973;Vervoort et al., 1986). An intermediate formed concurrently with light emission (IV) was isolated and characterized by Kurfiirst et al. (1984Kurfiirst et al. ( ,1987. However, an intermediate (111) postulated to occur upon reaction of I1 with aldehyde via the Michaelis complex (IIA), but preceding light emission, remained to be demonstrated.Since Kurfiirst et al. (1984) used decanal, a fast-reacting aldehyde, we thought it possible that the existence of this earlier intermediate might be easier to detect under conditions where the reaction proceeds more slowly. Accordingly, dodecanal and octanal were selected as aldehydes (Hastings et al., 1969), and experiments were performed at a lower temperature (-4 "C).The reactions were carried out starting with the luciferasebound flavin 4a-hydroperoxide (11) prepared and purified in advance. Concurrent measurements of absorption and light emission were made. The results show three sets of isosbestic points, indicating the existence of an additional intermediate, which is postulated to correspond to the luciferase flavin 4a- Abbreviations: FMN and FMNH2, riboflavin 5'-phosphate and its reduced form; NMR, nuclear magnetic resonance.

Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii

“…3 is likely to be C(4a)-hydroperoxy-FMN (1-3). Spectrum B is also similar to that of C(4a)-hydroperoxyflavins generally found in the class of single component aromatic hydroxylases (1, 3, 24 -26), as well as for luciferase (the first two-component flavin-dependent oxygenase studied in detail) (27,28), cyclohexanone monooxygenase (29) (32), and also greater than the rate of formation of the C(4a)-flavin hydroperoxide at 0.13 mM oxygen, 185 Ϯ 9 s Ϫ1 (Fig. 2).…”

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

“…The former case is observed in Baeyer-Villiger monooxygenases (such as cyclohexanone monooxygenase) (19) or bacterial luciferase (20) in which the flavin C4a-peroxide acts as a nucleophile attacking the substrate. On the contrary, a flavin C4a-hydroperoxide has been well documented in flavin-containing monooxygenases (21) and aromatic hydroxylases (22)(23)(24), in which the terminal oxygen of hydroperoxyflavin acts as an electrophile in an aromatic substitution reaction.…”

Bioluminescent bacteria: lux genes as environmental biosensors

“…The first step in the luminescence reaction pathway involves binding of a single FMNH 2 (20,21,58). Next, aldehyde reacts with EFHOOH to form a flavin-oxygen-aldehyde intermediate, also called a peroxyhemiacetal (E-FOOA) (31). This intermediate has not yet been isolated but it is believed that its lifetime is the rate-limiting step of the reaction.…”