Fluorescence energy transfer in tryptophanase

Excitation of apotryptophanase from Escherichia coli$\text{B}\text{lt7-A}$ at 290 nm yielded a fluorescence emission centered at 340 nm. Binding of pyridoxal phosphate to apoenzyme induced quenching of protein fluorescence concomitant with an appearance of another peak at 510 nm by way of energy transfer from tryptophan. Based on the results, an approximate distance between the coenzyme and tryptophan was estimated to be 18–24 Å according to the Förster's theory. The ozone-inactivated enzyme yielded only the 340 nm-peak upon excitation at 290 nm following reconstitution with the coenzyme. The fluorescence decay time of the tryptophyl residue was somewhat increased by ozone-inactivation. These results suggest that the tryptophyl residue essential for the activity is involved in a direct interaction with the coenzyme.     

Cooligopeptides containing aromatic residues spaced by glycyl residues. IX. Fluorescence properties of tryptophan-containing peptides

The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λ_max) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)_n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λ_max, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λ_max of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

Conformational characterization of human eukaryotic initiation factor 2α: A single tryptophan protein

The α-subunit of the human eukaryotic initiation factor 2 (heIF2α), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2α gets phosphorylated by eIF2α-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2α, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (τ₁ = 1.13 ns and τ₂ = 4.74 ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the α-helix and β-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted α-helix and rearranged β-sheet in presence of 4 M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 °C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Correlation between internal motion and emission kinetics of tryptophan residues in proteins

Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.     

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.     

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.     

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β-d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ_S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ_L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ_S and φ_L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ₁ = 6–7 ns, τ₂ = 2.0–2.3 ns, and τ₃ ≤ 0.1 ns (the native state), and τ₁ = 4.4–4.6 ns, τ₂ = 1.7–1.8 ns, and τ₃ ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.     

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

The fluorescence decay of tryptophan residues in native and denatured proteins.

The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment.     

Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.     

Rotational freedom of tryptophan residues in proteins and peptides

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.     

Phase fluorimetric lifetime spectra. I. In algal cells at 77 K

A new method for decomposing fluorescence emission spectra into their elementary components, based on the simultaneous recording of fluorescence intensity and lifetime vs. the emission wavelength, has been applied to the spectra of algal cells at liquid nitrogen temperature. A model of Gaussian components fits both τ(λ) and F(λ) spectra with the same parameters. The fluorescence lifetimes have been measured by phase fluorimetry at two modulation frequencies: 29 and 139 MHz. The final Gaussian decomposition is able to describe both the 29 and 139 MHz spectra. The following conclusions concerning the fluorescence spectra of Chlorella cells at 77 K can be drawn. These conclusions are also valid with minor changes for the other examined species. (1) An overlapping of different emitting bands occurs in all the spectra; therefore, a direct lifetime reading from phase delay measurement necessitates measurements being made at several frequencies. (2) At the F_max fluorescence level, the lifetime values of the two emissions usually associated with variable fluorescence are 0.53 ns (for B′₁; λ peak 688 nm), and 1.46 ns (for B′₂; λ peak 698 nm); these lifetimes are shorter than those we have measured at room temperature (approx. 1.8 ns). (3) Superimposed on B′₁ and B′₂ and with approximatively the same peak location, two long-lifetime components (B″₁, 4.8 ns; B″₂, 5.6 ns) are present. Two hypotheses can be proposed to explain these emissions: (i) the long-lifetime components arise from subsets of chlorophyll a disconnected from the functional antenna by the cooling process; and (ii) charge recombination in reaction centers leads to delayed fluorescence. (4) In the λ > 710 nm region, two main bands are required to describe the so-called Photosystem I emission: B₃ (0.8 ns; λ peak 715 nm) and B₄ (3.3 ns; λ peak 724 nm). The former band, usually unresolved in the amplitude fluorescence spectra, is a specific finding from lifetime measurements and has been associated with the antenna core of Photosystem I. No additional information has been obtained for B₄. A supplementary small band (B₅, 0.40 ns; $\text{λ}\text{peak}\text{? 740}\text{nm}$) is necessary to take into account the frequency effect and the τ(λ) decrease in the λ > 740 nm spectral range.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.     

Ferredoxin from Halobacterium of the Dead Sea. Structural properties revealed by fluorescence techniques

The two tryptophan residues of ferredoxin from Halobacterium of the Dead Sea differ in their fluorescence characteristics. One of these tryptophan residues (class 1) absorbs more to the red and is thus probably in a more apolar environment than the other (class 2). Upon removal of the ferric ions, i.e., in the apoferredoxin, a 2.2-fold increase in the quantum yield of fluorescence is observed. A double exponential decay of the fluorescence is found for ferredoxin, reduced ferredoxin, as well as for the apoferredoxin. The longer decay time assumes a constant value of 6.9 ns in all three cases, indicating that it originates in a tryptophan residue which is not affected by changes in the Fe³⁺ binding site (class 2 tryptophan). The shorter decay component increases gradually from 0.55 ns in oxidized ferredoxin, through 0.80 ns in the reduced ferredoxin to 1.24 ns in the apoprotein. This decay component is thus assumed to be largely due to the second tryptophan residue of the protein (class 1) located close to the Fe³⁺ binding site. On the other hand, the relative decay amplitude of the class 2 tryptophan is doubled upon formation of apoferredoxin. It is concluded that the class 1 tryptophan is quenched by the active site ferric ions and that the class 2 tryptophan is partially exposed to a polar environment. Whereas class 1 tryptophan may be similar to the single nonfluorescent tryptophan of spinach ferredoxin, class 2 tryptophan is found in a peptide which is present only in halophilic ferredoxins. Conformational changes occur in the molecule upon removal—but not reduction—of the ferric ions, causing the environment of the class 2 tryptophan to become more hydrophobic. It is possible that the class 1 tryptophan is associated with the occurrence of a higher redox potential in this ferredoxin, when compared with chloroplast-type ferredoxins.