Studies on the bacterial luciferase reaction: isotope effects on the light emission. Is a "CIEEL" mechanism involved?

Macheroux, Peter; Ghisla, Sandro; Kurfürst, Manfred; Hastings, J. Woodland

doi:10.1515/9783111521350-119

Cited by 8 publications

(4 citation statements)

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“…The exogenous imidazole in the αH44A system and the αHis44 residue in the native luciferase system could first abstract a proton from the flavin peroxyhemiacetal intermediate III at the boxed position shown in Scheme followed by a hydride transfer from the circled position according to a Baeyer−Villiger mechanism proposed some years ago () and resurrected recently (). Alternatively, the catalytic base could be involved in facilitating the transfer of a proton from the circled position following an electron/charge transfer (a modified version of the chemically induced electron exchange) mechanism ( − ). In either case, this proposed catalytic role is consistent with the known deuterium isotope effect ( D k = 1.4−1.7) of C1-deuterated aldehydes on luciferase bioluminescence decay rate ( 30 , 34 , 35 ).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Catalytic Base in Bacterial Luciferase by Chemical Rescue of a Dark Mutant

Huang

1997

Biochemistry

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Mutation of the His44 residue of the alpha subunit of Vibrio harveyi luciferase to an alanine was known to reduce the enzyme bioluminescence activity by five orders of magnitude [Xin, X., Xi, L., and Tu, S.-C. (1991) Biochemistry 30, 11255-11262]. We found that the residual activity of the alpha H44A luciferase was markedly enhanced by exogenously added imidazole and other simple amines. The peak luminescence intensity in nonturnover assays was linearly proportional to levels of alpha H44A and the rescue agent, indicating a lack of significant binding under our experimental conditions. The rescue effect of imidazole was pH dependent and quantitatively correlated well with the amount of imidazole base. The rescue efficiencies of imidazole and amines were found to be regulated by both their molecular volume and pKa. A Brønsted analysis revealed a beta value of 0.8 +/- 0.1. The enhancement of alpha H44A activity by imidazole took place after the formation of the flavin 4a-hydroperoxide intermediate. The predominant form of the flavin 4a-hydroperoxide intermediate generated by alpha H44A was inactive in bioluminescence, but was reactive with the aldehyde substrate for bioluminescence in the presence of imidazole. These findings, taken together, provide evidence for assigning a role for the alpha His44 imidazole as a catalytic base in the luciferase reaction. This study provides the first characterization of a catalytic residue for bacterial luciferase and the first demonstration of the rescue of a histidine-mutated enzyme by exogenous imidazole and amines.

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Catalytic Base in Bacterial Luciferase by Chemical Rescue of a Dark Mutant

Huang

1997

Biochemistry

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“…After a still not fully understood mechanism, intermediate III decays to form an exited enzyme-bound emitter. One proposed pathway involves the formation of a caged pair of a carboxylic acid radical anion and 4a-hydroxy-4a,5-dihydroFMN radical cation (HFOH +• ) intermediate IV + • ( − ). A subsequent radical annihilation generates a carboxylic acid and the excited state of 4a-hydroxy-4a,5-dihydroFMN (HFOH*) intermediate IV* as the emitter.…”

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confidence: 99%

Identity of the Emitter in the Bacterial Luciferase Luminescence Reaction: Binding and Fluorescence Quantum Yield Studies of 5-Decyl-4a-hydroxy-4a,5-dihydroriboflavin-5‘-phosphate as a Model

Lei

Ding

2004

Biochemistry

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The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.

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“…As introduced in the preceding review article [5], the recently proposed CIEEL mechanism (Chemically I•nitiated Electron Exchange Luminescence) [6] merits careful consideration. In preliminary ~experiments [7,8] aiming at its verification, we attempted a correlation of the rate of emission decay with the redox potential of flavins, substituted at various ring positions.…”

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confidence: 99%

“…alteration of protein-flavin interactions. It has been shown, using FMN analogues carrying various substituents at position C (8), that these are recognized by luciferase, and are competent in bioluminescence, suggesting that this position is "free", Le. exposed to solvents [9,10] that modifications restricted conserve intact flavin-protei prejudice the catalytic event.…”

mentioning

confidence: 99%

Studies on the mechanism of bacterial bioluminescence. Evidence compatible with a one electron transfer process and a CIEEL mechanism in the luciferase reaction

Macheroux

Eckstein

Ghisla

1987

Flavins and Flavoproteins 1987

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Studies on the bacterial luciferase reaction: isotope effects on the light emission. Is a "CIEEL" mechanism involved?

Cited by 8 publications

References 0 publications

Identification and Characterization of a Catalytic Base in Bacterial Luciferase by Chemical Rescue of a Dark Mutant

Identification and Characterization of a Catalytic Base in Bacterial Luciferase by Chemical Rescue of a Dark Mutant

Identity of the Emitter in the Bacterial Luciferase Luminescence Reaction: Binding and Fluorescence Quantum Yield Studies of 5-Decyl-4a-hydroxy-4a,5-dihydroriboflavin-5‘-phosphate as a Model

Studies on the mechanism of bacterial bioluminescence. Evidence compatible with a one electron transfer process and a CIEEL mechanism in the luciferase reaction

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