Bacterial luciferase, mechanism

The biochemical mechanism of bacterial luminescence has been studied in detail and reviewed by several authors (Hastings and Nealson, 1977 Ziegler and Baldwin, 1981 Lee et al., 1991 Baldwin and Ziegler, 1992 Tu and Mager, 1995). Bacterial luciferase catalyzes the oxidation of a long-chain aldehyde and FMNH2 with molecular oxygen, thus the enzyme can be viewed as a mixed function oxidase. The main steps of the luciferase-catalyzed luminescence are shown in Fig. 2.1. Many details of this scheme have been experimentally confirmed. 

Eckstein, J. W., and Ghisla, S. (1991). On the mechanism of bacterial luciferase. 4a,5-Dihydroflavins as model compounds for reaction intermediates. In Flavins Flavoproteins, Proc. Int. Symp., 10th, 1990, 269-272. 

Kaaret, T. W., and Bruice, T. C. (1990). Electrochemical luminescence with N(5)-ethyl-4a-hydroxy-3-methyl-4a,5-dihydrolumiflavin. The mechanism of bacterial luciferase. Photochem. Photobiol. 51 629-633. 

Meighen, E. A., and Bartlet, I. (1980). Complementation of subunits from different bacterial luciferases. Evidence for the role of the (3 subunit in the bioluminescent mechanism. J. Biol. Chem. 255 11181— 11187. 

Waddle, J. J., Johnston, T. C., and Baldwin, T. O. (1987). Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo. Biochemistry 26 4917-4921. 

At least five detailed mechanisms have been proposed for bacterial luciferase within the last six years (28, 47, 48, 49, 50). With the assumption that our biomimetic model relates to the enzyme-catalyzed reaction, we conclude that these five proposals of mechanisms are incorrect. Four (28, 47,48) are inconsistent with the intermediacy of the mixed peroxide (4a-FlEt—O—O—CH(OH)R ) and cannot be applied to N(5)-alkylflavins. In addition, two (28, 47) of these mechanisms as well as another (49), require the hydroxyl group of the aldehyde-peroxide adduct. No mechanism can be taken seriously without the identification of the excited species. We do know that a hydrogen substituted is required on the carbon that is converted to a carbonyl group (47). 

To estimate the contribution of the second mechanism the interaction of bioluminescent enzymes with halide-containing dyes was investigated. Two types of dyes were tested xanthene and anthracene derivatives. The interaction of dyes with bacterial luciferase and apo-obelin was studied. 

MECHANISM OF BACTERIAL LUCIFERASE ENERGETIC AND QUANTUM YIELD CONSIDERATIONS... 

The reaction mechanism of bacterial luciferase has been studied extensively.1 2 An electron-exchange reaction mechanism (Scheme l)3 4 has been postulated and gained considerable acceptance. 

Mechanism of Bacterial Luciferase Energetic and Quantum Yield Considerations 73... 

Scheme 26 CIEEL mechanism proposed for bacterial luciferase.

Fig. 180. Postulated reaction mechanism catalyzed by bacterial luciferase...

The foundation for the chemical mechanism of bacterial luciferase is primarily laid by the work of Hastings and colleagues. " These studies and the work of others were the subjects of a number of reviews. Main features of the luciferase chemical mechanism are summarized in Scheme 136.2. The luciferase-bound Nl-deprotonated FMNH" (Intermediate I) reacts with oxygen to generate the 4a-hydroperoxy-FMNH (HF-4a-OOH Intermediate II), which undergoes a dark decay to form FMN and HjOj in the absence of aldehyde or reacts with aldehyde to form the 4a-peroxyhemiacetalFMNH (Intermediate III). 

The kinetic mechanism will be confined to V. harveyi luciferase, the most extensively studied bacterial luciferase to date. Our version of the kinetic scheme is shown in Scheme 136.6. Aside fi-om the aldehyde... 

Macheroux, R, Ghisla, S., Kurfiirst, M., and Hastings, J.W., Studies on the bacterial luciferase reaction isotope effects on the Hght emission. Is a CIEEL mechanism involved , in Flavins and Flavoproteins, Bray, R.C., Engel, PC., and Mayhew, S.G., Eds., Walter deGruyter, Berlin, 1984, p. 669. 

Francisco, W.A., Abu-Soud, H.M., DelMonte, A.J., Singleton, D.A., Baldwin, TO., and Raushel, F.M., Deuterium kinetic isotope effects and the mechanism of the bacterial luciferase reaction. 

Holzman, T.F. and Baldwin, T.O., Reversible inhibition of the bacterial luciferase catalyzed bioluminescence reaction by aldehyde substrate kinetic mechanism and Ugand effects. Biochemistry, 22, 2838, 1983. 

Fig. 2.1 Mechanism of the bacterial bioluminescence reaction. The molecule of FMNH2 is deprotonated at N1 when bound to a luciferase molecule, which is then readily peroxidized at C4a to form Intermediate A. Intermediate A reacts with a fatty aldehyde (such as dodecanal and tetradecanal) to form Intermediate B. Intermediate B decomposes and yields the excited state of 4a-hydroxyflavin (Intermediate C) and a fatty acid. Light (Amax 490 nm) is emitted when the excited state of C falls to the ground state. The ground state C decomposes into FMN plus H2O. All the intermediates (A, B, and C) are luciferase-bound forms. The FMN formed can be reduced to FMNH2 in the presence of FMN reductase and NADH.

Eckstein JW, Hastings JW, Ghisla S. Mechanism of bacterial bioluminescence 4a,5-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substitutions at the 8-position of flavin on luciferase kinetics. Biochemistry 1993 32 404-11. 

The mechanisms of inhibition/stimulation of bacterial luminescence are not fully understood. A luciferase-flavin adduct is formed in an excited state and the excess of energy is then released in the form of visible light (Nakamura et al. 1982). Any substance able to interfere in one or more steps of this reaction, as shown by reduced light emission, is classified as toxic. Also, a substance which may interact with any of the other biochemical functions of the bacteria, resulting in the death or a reduced metabolic activity of the organisms is... 

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