Biolumineszenz und Blaulichtwahrnehmung
Die Biolumineszenz und die Absorption von "Blaulicht" sind zwei extrem verbreitete Ph¿nomene, deren molekulare Grundlagen noch weitgehend unbekannt sind. Bei der Biolumineszenz wird chemische Energie in Licht umgewandelt. Die Lichtemission hat eine Vielfalt von Funktionen wie z.B. Beeinflussung des Sexualverhaltens, Nahrungssuche, Verteidigung usw. Blaulicht wird von den meisten höheren
Organismen erkannt und diese Funktion dient z.B. der Steuerung des Wachstums (Pflanzen) oder der Synchronisierung der "inneren Uhr" (circardian rhytm). Wir untersuchen einige dieser Phänomene bei denen Flavoproteine entweder als Licht-Emitter oder als -Prim¿rrezeptoren eine Rolle spielen. Im Falle der bakteriellen Luciferase untersuchen wir den Mechanismus der Oxidation von langkettigen
Aldehyden durch (enzymgebundene) Flavinhydroperoxyde und die damit gekoppelte Erzeugung von Licht. Im Falle der "Blaulicht"-Absorption haben wir ein neues Projekt in Angriff genommen. Dessen Ziel ist die Expression des Blaulichtrezeptors in E. coli und zunächst die Charakterisierung der darin enthaltenen Chromophore, letztere leiten sich vermutlich von Flavinen und Pteridinen ab. In einer zweiten Phase soll die Umwandlung der Prim¿ranregung in für die Zelle verwertbare Signale untersucht werden. (Kooperation mit Prof. A. Batschauer, Marburg).
The bioluminescence and the absorption of blue light are two wide-spread phenomena. In spite of this
our understanding of their molecular basis ist still very poor. In the case of thebioluminescence chemical
energy is transformed into light and the emission of the latter has an array of functions such as the
regulation of sexual behavior, the collection of food, or defence. A process in the opposite direction is
observed in the "blue-light" phenomenon where light is transformed into a signal which can be used by
the cell for a variety of functions. Examples are the regulation of growth in plants and the synchronisation
of the circardian rhytm in higher organisms. We investigate some of these phenomena focussing on
those in which flavins and flavoproteins play a role. With bacterial luciferase we study the conversion of
energy, which arises from the oxidation of long chain aldehydes by a flavin-hydroperoxide, into light. In
the case of the "blue-light" receptors we want to express the primary receptor in <I>E. coli</i> and to
characterize its chromophores, probably flavins and pterins. In a second stage we want to study the
transformation of the signal arising from the primary excitation into a signal which can be understood by
- FB Biologie
|(1994): Bacterial luciferase : bioluminescence emission using lumanzines as substrates Flavins and flavoproteins 1993 : proceedings of the eleventh International Symposium Nagoya (Japan), July 27 - 31, 1993 / Yagi, Kunio et al. (Hrsg.). - Berlin [u.a.] : de Gruyter, 1994. - S. 839-842. - ISBN 3-11-014165-5|
Macheroux, Peter; Ghisla, Sandro; Hastings, J. Woodland
|(1993): Mechanism of bacterial bioluminescence : 4a,5-Dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics Biochemistry ; 32 (1993), 2. - S. 404-411. - ISSN 0006-2960. - eISSN 1520-4995|
Mechanism of bacterial bioluminescence : 4a,5-Dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics
Bioluminescence catalyzed by bacterial luciferases was measured using FMN, iso-FMN (6-methyl-8-nor-FMN), and FMN analogs carrying the following substituents at position 8: -H, -Cl, -F, SMe, SOMe, S02Me, or -0Me. The first-order rate constants for the decay of light emission correlate with the one-electron oxidation potentials of the 4a,5-dihydro forms of the FMN analogs. To determine thevalues of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydros tructure and -H, -CH3, -C1, -OCH3, and -NH2 as substituents at position 8 have been synthesized as models for the 4a-peroxy-4a,5-dihydroflavin intermediates occurring during catalysis by the flavin-dependent monooxygenase luciferase. The tetrahydropyrrole ring between positions 4a and 5 of these isoalloxazine derivatives stabilizes the 4a,5-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials (Eobs) were measured by cyclic voltammetry and used to determine the empirical coefficients in the Swain equation. On the basis of this, the one-electron oxidation potentials of 4a,5-propano-4a,5-dihydraon alogs with other substituents in position 8 were calculated (Ecalc). The bioluminescence reaction rate is fastest with FMN analogs of lowest oxidation potential; Le., the slope of the correlation is negative. This indicates that in the rate-limiting step the 4a,5-dihydroflavin moiety donates negative charge. The results are compatible with an intramolecular, chemically initiated electron exchange luminescence mechanism for the bacterial luciferase reaction. A good correlation was also found between EOba nd the literature values of the 2-electron oxidation/reduction potentials (Eredox) of the couple FIox/1,5-dihydro-FIrf~o r the flavin derivatives having the same substituent at position 8. The effects of the substituent in position 8 on the redox properties of 1,5-dihydro- and 4a,5-dihydroflavins are thus essentially the same. This indicates that the earlier use of readily available redox potentials for FIox/1,5-dihydro-FIred for studying reactions involving the 4aS-dihydro isomer was sound.
|(1993): Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction Biochemistry ; 32 (1993), 51. - S. 14183-14186. - ISSN 0006-2960. - eISSN 1520-4995|
Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction
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 °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.
|(1990): A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system : energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1 Proceedings of the National Academy of Sciences of the United States of America ; 87 (1990), 4. - S. 1466-1470|
A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system : energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1
Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio fischeri strain Y-1b. Its presence in a luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 184.108.40.206] reaction mixture causes a striking color change, and an increase in bioluminescence intensity, as well as a faster rate of intensity decay, so that the quantum yield is not changed. The emission spectrum shows two distinct color bands, one at 490 nm attributed to the unaltered emission of the luciferase system, the other peaking in the yellow around 540 nm due to YFP emission. The kinetics of the two color bands differ, so the spectrum changes with time. The yellow emission reaches its initial maximum intensity later than the blue, and then both blue and yellow emissions decay exponentially with nearly the same pseudo-first-order rate constants, linearly dependent on [YFP] (from 0.01 sec-1 with no YFP to a maximum of ≈0.1 sec-1 at 4°C) but exhibiting a saturation behavior. The data can be interpreted by assuming the interaction of YFP with the peroxyhemiacetal intermediate in the luciferase reaction to form an unstable new complex whose breakdown gives the yellow emitter in its excited state. This simple model fits well the data at [YFP] < 15 µM. The results indicate that a single primary excited state cannot be responsible for the blue and the yellow emissions.
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