Interaction of the ground and excited states of indole derivatives with electron scavengers

Steiner, Robert F.; Kirby, Edward P.

doi:10.1021/j100846a015

Cited by 186 publications

(110 citation statements)

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“…Several explanations are plausible. It is noteworthy that cysteine has been reported to act as an efficient dynamic quencher of several types of fluorophores (Steiner & Kirby, 1969;Harris & Hudson, 1990), and mutation of Lys-58 to Cys may simply provide a thiol quencher near the adenylyl group. This does not seem likely because the side chain of Lys-58 appears to point toward the active site, away from the site of adenylylation.…”

Section: Discussionmentioning

confidence: 99%

Time‐resolved fluorescence and computational studies of adenylylated glutamine synthetase: Analysis of intersubunit interactions

et al. 1993

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Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo. The overall structure of the enzyme consists of 12 subunits arranged as two hexamers, face to face. Research reported in this paper addresses the question of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon. Wild-type GS has two Trp residues (positions 57 and 158) and the adenylylation site lies within 7-8 A of the Trp-57 loop in the adjacent subunit of the same hexameric ring; Trp-158 is about 35 A from the site of adenylylation. Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS. One fluorophore is E-AMP adenylylated GS (at Tyr-397), and the other fluorophore is the intrinsic protein residue Trp-57. These experiments were conducted in order to detect possible intersubunit interactions between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS. The fluorescence due to E-AMP of two adenylylated enzymes, wild-type GS and the W158F mutant, exhibits heterogeneous decay kinetics; the data adequately fit to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively. The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emission wavelengths. The WS~L-E-AMP enzyme yields longer average lifetime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component. An additional residue in the Trp-57 loop, Lys-58, has been altered and the K58C mutant enzyme has been adenylylated with &-AMP on Tyr-397. Lys-58 is near the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS. The fluorescence of E-AMP-adenylylated K58C mutant GS is best described by a triple exponential decay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, with the largest fraction being the median lifetime component. Relative quantum yields of ~-AMP-Tyr-397 were measured in order to determine if static quenching occurs from adenine-indole stacking in the wild-type GS. The relative quantum yield of the E-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predicted from the difference in lifetime values: thus, no static quenching is evident. Intrinsic tryptophan fluorescence was also studied in the presence and absence of covalently attached adenylyl groups. The fluorescence decay parameters of Trp-57 are not significantly affected by the presence of E-AMP or AMP attached to Tyr-397. Enzymatic activity of the mutant proteins was also studied. The Mg-activated enzymes are nearly completely inhibited upon adenylylation, and regulation is not affected by mutation at Trp-57 or Lys-58. These results indicate that the Trp-57 loop dynamically interacts with the adenylyl group, but a "ring stacked" complex is not f...

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Section: Discussionmentioning

confidence: 99%

Time‐resolved fluorescence and computational studies of adenylylated glutamine synthetase: Analysis of intersubunit interactions

et al. 1993

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“…reductant, and that triplet excited riboflavin can oxidize ground state indole. Steiner and Kirby (1969) compared rates of quenching of acetyltryptophan fluorescence by oxidants with their ability to scavenge electrons. An electron transfer mechanism was suggested.…”

Section: Other Photoactive Molecules Of Biological Interestmentioning

confidence: 99%

The Energetics of Electron‐transfer Reactions of Chlorophyll and Other Compounds*

Seely

1978

Photochem & Photobiology

164

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Abstract-Quite often the primary photochemical reaction of an excited state molecule is transfer of an electron to or from another molecule in its ground state. Rates of such reactions are closely dependent on differences between ground and excited state redox potentials of the reagents. The solvent also plays an important role in stabilizing ion pairs formed by the electron transfer. This Review discusses experimental data relating rates to electrochemical energy parameters in the context of a scheme which portrays the energy and electron transactions in a unified manner. Three consequences of reaction of a singlet excited state are distinguished: (Sl) quenching without detectable products, (S2) exciplex fluorescence, (S3) transient radical ion production, and energetically necessary conditions are derived for each. Similarly, four kinds of reactions involving the triplet state are distinguished, which depend on the relation between the energy of the triplet state and that of the ion pair states: (TI) rapid quenching, (T2) slow quenching, (T3) accelerated intersystem crossing and (T4) generation by reaction between radical ions 'of like spin. The last may be followed by electrochemiluminescence. Classes of compounds for which data are available include chlorophylls, porphyrins and a few other molecules of biological interest, aromatic hydrocarbons and their derivatives, heterocyclic systems, carbonyl compounds, dyes, and complexes of Ru and U. A Table compiling median or selected values of ground and excited state electrochemical potentials of chlorophylls, some porphyrins, and a few other compounds is presented.

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“…This data shows that with increasing hydrogen ion concentrations corresponding to pH values below -3 intensities decrease (reversibly) more rapidly than expected from the titration of a single group. This suggests that the increasing free proton concentration is exerting a quenching effect, by perturbing the indole in the excited state either by binding of the proton to the excited indole or by abstracting an electron from the excited indole (19)(20)(21). To circumvent this complication, we titrated a control tryptophan-containing compound with no dissociable groups-namely, N-acetyl-L-tryptophan ethyl ester-and observed a qualitatively similar quenching with increasing H+ concentration.…”

mentioning

confidence: 99%

“…Support for this idea is that emission is reduced when the water solvent is replaced by isobutanol, even though the replacement might have been expected to suppress dielectric relaxation. Another widely considered and plausible mechanism is quenching by the transport of charge-protons through space and electrons through bonds or through space (19,20,22). For example, in tryptophan itself a proton from the a-amino group might be donated to the C-4 of the indole [the work of Barkley and her associates (23,24) has forced a change in this simplest hypothesis; it must now be surmised that a protonated carboxyl or amino group closely approaches the C-4 locus (25), enabling a proton or an electron to move through space to effect the quenching].…”

mentioning

confidence: 99%

On how a myosin tryptophan may be perturbed.

Bivin

Kubota²,

Pearlstein³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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A weil-known indication that a nucleotide has bound to myosin Is the enhancement of the fluorescence of a speciflc tryptophan in the "subfragment 1" segment of the protein. Empirically the effect has been enormously useful in myosin enzymology. But beyond an early suggestion that it aries from a purine-tryptophan charge-transfer complex, the mechanism of the effect has not been considered. Here we consider the alternative that it arises from an Ionizable group (either another residue or the phosphate of the nucleotide) whose proimit to the tryptophan is altered by substrate bnding. We study this possibility by studying the interaction of an ionizable residue and tryptophan when both are incorporated in a diketopiperazine structure. The geometry of the ituation is inferred from molecular mehaics simulations. Unexpectedly, the best explanation seems to be that the field of the imposed charge, acting across space, affects events in the excited state of the indole.The perturbation of the absorbance (1) and emission (2) (8), but alternative explanations have not been considered. We undertook the present study, hoping to get clues about the aforementioned effect in myosin and, more generally, information about environmental effects on tryptophan. Our specific goal was to study the effect of a single charge positioned in a known relation to the indole ring, using a setup in which the sign and quantity of the charge (and to a limited extent the geometry of the arrangement) could be varied. We sought to accomplish this goal by using diketopiperazines constructed from. tryptophan and the L or D forms of amino acids such as histidine or aspartic acid, using appropriate molecular mechanical analysis to characterize the charge-indole relation. MATERIALS AND METHODSPreparation of the Diketopiperazines. The strategy followed was to prepare t-butoxycarbonyl (Boc)-protected amino acids, to couple these into linear dipeptide esters, and then to cyclize them and remove the protective groups.Boc-Trp and Boc-Asp(13-OBz) (where Bz = benzoyl) were prepared according to Morodes et al. (9). Glu(-OBz) OBz-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6791TosOH and His-OMe*2HCl were prepared according to Zervas et al. (10) and Humphlett and Wilson (11), respectively. Linear protected dipeptide esters were prepared by the N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide method (12). Dimethylformamide was the solvent for coupling. The products were purified from ethyl acetate petroleum ether, except Boc-L-Trp-L-His-OMe, which was purified from warm ethanol. In TLC, each of the products gave a single spot (ninhydrin negative) using the KI/toluidine reaction for detection (13). To prepare the diketopiperazines, a Boc group was removed by dissolving 10 mmol of Bocprotected dipeptide ester in 120 ml (240 ml for the histidinecontaining dipeptide) of 0.1 M HCl/96% formic acid...

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Interaction of the ground and excited states of indole derivatives with electron scavengers

Cited by 186 publications

References 2 publications

Time‐resolved fluorescence and computational studies of adenylylated glutamine synthetase: Analysis of intersubunit interactions

Time‐resolved fluorescence and computational studies of adenylylated glutamine synthetase: Analysis of intersubunit interactions

The Energetics of Electron‐transfer Reactions of Chlorophyll and Other Compounds*

On how a myosin tryptophan may be perturbed.

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