Fluorescence quenching of Trp-314 of liver alcohol dehydrogenase by oxygen

The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.     

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.     

Room temperature phosphorescence of Trp-314 as a monitor of subunit communications in alcohol dehydrogenase from horse liver

The phosphorescence properties of liver alcohol dehydrogenase from horse were characterized at limiting concentrations of coenzyme and coenzyme analogues. The emission decay kinetics of Trp-314 in strong, slowly exchanging, ternary complexes with NADH/isobutyramide, NAD/pyrazole, and NADH/dimethyl sulfoxide displays a markedly nonexponential character. The analysis of decay components over the saturation curve reveals that the phosphorescence from singly bound protein molecules has a lifetime from 1 to 1.3 s, which is 2-3 times larger than observed with fully bound and unliganded enzyme. The remarkably tighter configuration reported by the triplet probe for the coenzyme-binding domain in half-saturated macromolecules is not exclusive of strongly inhibited ternary complexes. Measurements on binary complexes with NADH, ADPR, and the inactive coenzyme analogue 1,4,5,6-tetrahydronicotinamide adenine dinucleotide confirm that binding of the ligand to one subunit has qualitatively the same influence on protein structure. If the lifetime of Trp-314 provides clear evidence for an appreciable change in conformation at half-binding that is apparently triggered by the ADPR fragment of the coenzyme, such communication between subunits does not lead to allosteric phenomena in coenzyme binding.     

Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1

We examined the frequency-domain intensity decays of the intrinsic tryptophan fluorescence (Trp-59) from ribonuclease T1 (EC 3.1.27.3) (RNAase T1). At pH 5.5 in the native state (below 30 degrees C), the intensity decay of the single tryptophan residue is a single-exponential process. Conditions which result in protein unfolding were found to induce more complex intensity decays. At temperatures above 40 degrees C, or in the presence of guanidine hydrochloride, the intensity decays became obviously double exponential. In general, the main effect of temperature or guanidine was to induce a second subnanosecond component in the intensity decay. The increased complexity of the decays could not be explained by a unimodal distribution of decay times. These results indicate that conformational dispersion of protein structure can be one origin of the multi-exponential decays which are generally observed for protein fluorescence.     

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.     

Characterization of the AP endonucleases from Thermoplasma volcanium and Lactobacillus plantarum: Contributions of two important tryptophan residues to AP site recognition

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3'-5'exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.     

Parvalbumin conformers revealed by steady-state and time-resolved fluorescence spectroscopy

The binding of calcium to whiting (one tryptophan residue) and pike (one tyrosine residue) parvalbumins has been studied by means of kinetic and steady-state fluorescence techniques. The decay curves of the tryptophan and tyrosine fluorescence of the parvalbumins are best fitted by a sum of two exponents for any metal state of the proteins. The data can be interpreted as a nonexponential decay of the fluorescence of a single-type chromophore or in terms of equilibria between compact and relaxed conformers of the parvalbumins in each metal state. Fluorescence quenching by I-ions and effects of H2O/D2O substitution confirm the second interpretation. The constants of the equilibria have been evaluated.     

Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.

The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.     

Phosphorescence properties and protein structure surrounding tryptophan residues in yeast, pig, and rabbit glyceraldehyde-3-phosphate dehydrogenase

An investigation of the phosphorescence emission properties of tryptophan (Trp) was carried out in glyceraldehyde-3-phosphate dehydrogenase from yeast and from pig and rabbit muscle. Aided by the external heavy-atom effect of iodide, the dependence on excitation wavelength, and thermal quenching profiles, it was established that the 0,0 vibronic band peaked at 406 nm in the pig and rabbit proteins is made up of overlapping contributions from two Trp residues. In contrast to a previous report [Davis, J.M., & Maki, A.H. (1984) Biochemistry 23, 6249-6256], this implies that even in the muscle enzymes all three aromatic side chains are phosphorescent. Further, when the nature of the local environment of each residue is compared to the crystallographic structure of lobster GPDH, it leads to a complete new assignment of the individual phosphorescence spectra. With each protein, a single Trp, identified as Trp-310, was found to display long-lived phosphorescence at room temperature. The decay of this emission gives evidence of conformational homogeneity among the subunits of the tetrameric molecule.     

Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state

Information on the effects of crystallization upon the structure of liver alcohol dehydrogenase from horse is obtained from a comparison of the phosphorescence properties of its tryptophan residues in solution and in the crystalline state. In the crystalline state the red shift in the phosphorescence spectrum of the solvent-exposed Trp-15 attests to a decreased polarity of its environment consistent with its shielding away from the aqueous solvent probably through its involvement in an intermolecular contact. On the other hand, the triplet-state lifetime of Trp-314 which is buried deeply in the coenzyme-binding domain demonstrates that the flexibility of this region of the macromolecule is unaffected by crystallization; a conclusion supported also by the similarity in the rate of oxygen quenching of its phosphorescence. Given that lattice constraints strongly inhibit large-scale conformational changes these results allow us to identify the average solution structure with the 'open' conformer determined crystallographically.     

Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one-photon and two-photon excitation.

We measured the emission spectra, intensity decays and anisotropy decays of the single tryptophan residue of human serum albumin (HSA) resulting from one-photon (295-298 nm) and two-photon (590-596) excitation. The emission spectra and intensity decays were independent of the mode of excitation. The anisotropy decays were superficially similar for one- and two-photon excitation. However, upon consideration of the different orientation photoselection for one- and two-photon excitation, the anisotropy data reveal different angles between the absorption and emission oscillators for one-photon and two-photon excitation. This result suggests different relative one-photon and two-photon cross-sections for the 1La and 1Lb transitions of the indole residue. This first report of the time-resolved anisotropy decay of a protein resulting from two-photon excitation suggests that such measurement will yield insights into the complex photophysical properties of tryptophan residues in proteins.     

Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme.

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.     

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.     

Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution

The extent of fluorescence quenching and that of phosphorescence quenching of Trp-15 and Trp-314 in alcohol dehydrogenase from horse liver as well as the intrinsic phosphorescence lifetime of Trp-314 in fluid solution have been utilized as structural probes of the macromolecule in binary and ternary complexes formed with coenzyme, analogous, and various substrate/inhibitors. Luminescence quenching by the coenzyme reveals that (1) while the reduced form quenches Trp emission exclusively from the fluorescent state, the oxidized form is very effective on the phosphorescent state as well and that (2) among the series of NADH binary and ternary complexes known by crystallographic studies to attain the closed form, distinct nicotinamide/indole geometrical arrangements are inferred from a variable degree of fluorescence quenching. Information of the dynamic structure of the coenzyme-binding domain derived from the phosphorescence lifetime of Trp-314 points out that within the series of closed NADH complexes there is considerable conformational heterogeneity. In solution, the variability in dynamical structure among the various protein complexes emphasizes that the closed/open forms identified by crystallographic studies are not two well-defined macrostates of the enzyme.     

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.     

Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue

The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the alpha-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (delta W3) or 31 (delta W31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (tau approximately 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to approximately 50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and delta W31 mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II)

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.