Fluorescent Polyene Aliphatics as Spectroscopic and Mechanistic Probes for Bacterial Luciferase: Evidence Against Carbonyl Product From Aldehyde as the Primary Excited Species

Cho, Ki-Woong; Tu, Shiao‐Chun; Shao, Ruxin

doi:10.1111/j.1751-1097.1993.tb02308.x

Cited by 9 publications

(6 citation statements)

References 33 publications

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“…Absorption spectrum of YFP and emission spectra of luciferase bioluminescence and YFP fluorescence. The corrected in vitro bioluminescence spectrum was taken from the report by Cho et al (12)…”

Section: Resultsmentioning

confidence: 99%

Energy Transfer Evidence for In Vitro and In Vivo Complexes of Vibrio harveyi Flavin Reductase P and Luciferase ¶

Low¹,

Tu²

2007

Photochemistry and Photobiology

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Conservation of energetically “expensive” metabolites is facilitated by enzymatic intra‐ and intermolecular channeling mechanisms. Our previous in vitro kinetic studies indicate that Vibrio harveyi reduced nicotinamide adenine dinucleotide phosphate–flavin mononucleotide (NADPH‐FMN) oxidoreductase flavin reductase P (FRP) can transfer reduced riboflavin 5′‐phosphate (FMNH2) to bacterial luciferase by direct channeling. However, no evidence has ever been reported for such an FMNH2 channeling between these two enzymes in vivo. The formation of a donor–acceptor enzyme complex, stable or transient, is mandatory for direct metabolite channeling between two enzymes regardless of details of the transfer mechanisms. In this study, we have obtained direct evidence of in vitro and in vivo FRP–luciferase complexes that are functionally active. The approach used is a variation of a technique previously described as Bioluminescence Resonance Energy Transfer. Yellow fluorescence protein (YFP) was fused to FRP to generate an active FRP–YFP fusion enzyme, which emits fluorescence peaking at 530 nm. In comparison with the normal 490 nm bioluminescence, an additional 530 nm component was observed in both the in vitro bioluminescence from the coupled reaction of luciferase and FRP–YFP and the in vivo bioluminescence from frp gene–negative V. harveyi cells that expressed FRP–YFP. This 530 nm bioluminescence component was not detected in a control in which a much higher level of YFP was present but not fused to FRP. Such findings indicate an energy transfer from the exited emitter of luciferase to the FRP component of the luciferase–FRP–YFP complex. Hence, the formation of an active complex of luciferase and FRP–YFP was detected both in vitro and in vivo.

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Section: Resultsmentioning

confidence: 99%

Energy Transfer Evidence for In Vitro and In Vivo Complexes of Vibrio harveyi Flavin Reductase P and Luciferase ¶

Low¹,

Tu²

2007

Photochemistry and Photobiology

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“…Stronger support for this idea would be with more efficient acceptors achieved by having them clearly bound to the Lux. Although the parinaldehydes gave a negative result , an aliphatic aldehyde with an attached fluorophore having absorbance in the range 350–400 nm should work, or alternatively a fluorophore conjugated to the Lux, both of these should produce a complete shift as seen with the natural sensitizers LumP and YFP. That such an alternate proximate acceptor sensitization is feasible is demonstrated with a fusion Lux‐GFP that produced an orange bioluminescence color .…”

Section: Final Thoughtsmentioning

confidence: 99%

“…Cho and coworkers used parinaldehydes, long‐chain aliphatic aldehydes with a fluorescent moiety at the tail end and found no alteration of the normal bioluminescence spectrum. They concluded that this eliminated any excited carbonyl product playing the role of the HEI.…”

Section: Introductionmentioning

confidence: 99%

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

Lee

Müller²,

Visser

2018

Photochem & Photobiology

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After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.

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“…The primary emitting product has been postulated to be the excited singlet-state of the luciferase-hydroxyflavin complex (luciferase-FMNH-OH) formed via the breakdown of the luciferase-flavin peroxyhemiacetal intermediate (luciferase-FMNH-OO-CHOHR). [4][5][6][7][8] The recent results of Lei et al 9 from their study using a new model flavin compound demonstrate clearly that luciferase-hydroxyflavin is the primary emitting product.…”

Section: Introductionmentioning

confidence: 98%

Relationship between the redox change in yellow fluorescent protein of Vibrio fischeri strain Y1 and the reversible change in color of bioluminescence in vitro

Karatani

Izuta

Hirayama

2007

Photochem Photobiol Sci

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To elucidate the reversible change in the color of bioluminescence (BL) arising from Vibrio fischeri Y1, the relationship between the BL color and the redox state of endogenous yellow fluorescent protein (YFP), carrying riboflavin 5'-phosphate (FMN), has been investigated in vitro. YFP lost fluorescence with a maximum at 538 nm when reduced, and retrieved its original fluorescence upon reoxidation. Such a change in YFP fluorescence was analogous to that of free FMN. In the NADH/FMN oxidoreductase-coupled luciferase reaction with YFP, yellow BL peaking around 535 nm was largely depressed when sodium dithionite was added. This phenomenon can be attributed to the reduction of YFP; i.e., reduced YFP does not participate in the luciferase reaction as a secondary emitter. On admitting air into the reaction mixture, the yellow light characteristic of V. fischeri Y1 BL was regenerated. These results indicate that the reversible change in YFP fluorescence is caused by the redox change of YFP-bound FMN, and that the change in BL color between blue and yellow is associated with the redox state of YFP.

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Fluorescent Polyene Aliphatics as Spectroscopic and Mechanistic Probes for Bacterial Luciferase: Evidence Against Carbonyl Product From Aldehyde as the Primary Excited Species

Cited by 9 publications

References 33 publications

Energy Transfer Evidence for In Vitro and In Vivo Complexes of Vibrio harveyi Flavin Reductase P and Luciferase ¶

Energy Transfer Evidence for In Vitro and In Vivo Complexes of Vibrio harveyi Flavin Reductase P and Luciferase ¶

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

Relationship between the redox change in yellow fluorescent protein of Vibrio fischeri strain Y1 and the reversible change in color of bioluminescence in vitro

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