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

Macheroux, Peter; Eckstein, Jens; Ghisla, Sandro

doi:10.1515/9783110884715-104

Cited by 5 publications

(1 citation statement)

References 8 publications

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“…Equations 1 to 5 are drawn from the extensive literature on the biochemistry of bacterial luciferase (1,16,17,23,31,56). The exact mechanisms of light emission in this and related models, as well as the precise identity of the emitter, are still the subjects of discussion and research (1,28,33,34). However, these issues have little effect on the discussion presented here.…”

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confidence: 99%

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Blouin¹,

Walker²,

Smit³

et al. 1996

Appl Environ Microbiol

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Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.

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confidence: 99%

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Blouin¹,

Walker²,

Smit³

et al. 1996

Appl Environ Microbiol

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Mechanisms of flavoprotein‐catalyzed reactions

Ghisla

Massey

1989

European Journal of Biochemistry

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540

349

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Flavoproteins are a class of enzymes catalyzing a very broad spectrum of redox processes by different chemical mechanisms. This review describes the best studied of these mechanisms and discusses factors possibly governing reactivity and specificity.A large number of flavin-containing enzymes, several hundreds, has been uncovered to date. An unusual feature of flavoproteins is the variety of the catalytic reactions performed, which range from typical redox catalysis such as the dehydrogenation of an amino acid, or the activation of dioxygen, to photochemistry; from 'DNA damage repair' to light emission. These few examples illustrate the fact that the same coenzyme is able to catalyze, or take part in catalytic events which must vary widely from a mechanistic point of view. The chemistry underlying the conversion itself will be quite different from case to case. This versatility sets flavoproteins apart from most other cofactor-dependent enzymes, which, in general, each catalyze a single type of chemical reaction. The activation or 'steering' of a particular activity of the flavin results from the interaction with the protein at the active center. On the other hand, for the vast majority of these enzymes a common feature exists, that at some stage during the catalytic event a transfer of electrons takes place between the substrate and the flavin itself.The purpose of the present review is not to enumerate the different functions of flavoproteins, this having been done elsewhere [l -41, but to focus on the mechanisms of catalysis, and on the possible ways of interaction of the flavin nucleus with the protein, i.e. on how this brings about chemistry appropriate to the particular enzyme reaction. In order toCorrespondence to S. Ghisla,

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ELECTROCHEMICAL LUMINESCENCE WITH N(5)‐ETHYL‐4a‐HYDROXY‐3‐METHYL‐4a, 5‐DIHYDROLUMIFLAVIN. THE MECHANISM OF BACTERIAL LUCIFERASE

Kaaret

Bruice

1990

Photochem & Photobiology

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It has been proposed in the literature that the chemiluminescence of the flavoenzyme of bacterial luciferase is caused by a chemically initiated electron-exchange luminescence mechanism which provides an excited 4a-hydroxy-4a,5-dihydroflavin ([4a-FlHOH]*) as product of 1e- reduction of the radical 4a-FlHOH+.. Electrochemical/photon counting experiments were performed to assess the feasibility of this proposal. Potentials for step-wise oxidation of N(5)-ethyl-4a-hydroxy-4a,5-dihydroluminflavin (4a-FlEtOH) have been determined in dry N,N-dimethylformamide (DMF). Photon counting was carried out during the 1e- reduction of 4a-FlEtOH+.in both DMF and acetonitrile by use of an apparatus consisting of a photocell mounted below a Pt ring-disk electrode. By use of the ring-disk electrode a steady state concentration of [4a-FlEtOH]* could be maintained by continuous 1e- oxidation of 4a-FlEtOH----4a-FlEtOH+.and 1e- reduction of 4a-FlEtOH+.----4a-FlEtOH. A maximum of 14% collection (theoretical maximum is 18%) of FlEtOH.+ at the ring electrode was obtained below 5000 rotations per minute. Calibration of the apparatus using 9,10-diphenylanthracene allowed approximation of the quantum yield for 1e- reductive capture of 4a-FlEtOH+.as 10(-6) to 10(-4) in DMF and 10(-7) to 10(-5) in acetonitrile. No fluorescence for 4a-FlEtOH in DMF could be observed; if fluorescent, the efficiency of 4a-FlEtOH can be no greater than approximately 3 x 10(-5). No electrogenerated chemiluminescence is observed on the electrochemical recycling of FlEt+----FlEt2+ and FlEt2+----FlEt+.

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

Cited by 5 publications

References 8 publications

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Mechanisms of flavoprotein‐catalyzed reactions

ELECTROCHEMICAL LUMINESCENCE WITH N(5)‐ETHYL‐4a‐HYDROXY‐3‐METHYL‐4a, 5‐DIHYDROLUMIFLAVIN. THE MECHANISM OF BACTERIAL LUCIFERASE

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