Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin.

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems.     

Migration of small molecules through the structure of hemoglobin: evidence for gating in a protein electron-transfer reaction

It has previously been shown that the rates and activation energies for migration molecules of different sizes through myoglobin are very similar. The results were interpreted in terms of conformational changes in the protein structure that facilitate the passage of the different molecules to a similar extent. Here we ask whether the quaternary structural changes that accompany the binding of ligands (O2 or CO) to hemoglobin might influence the migration rate from the solution into the protein's binding site. As a model for the R state of hemoglobin, we used the protein in which the Fe protoporphyrin (FePP) in the alpha subunit was substituted by Zn protoporphyrin (ZnPP) and the oxidized heme was ligated by CN-. The T state of hemoglobin was represented by the protein in which all four FePP groups were substituted by ZnPP. The quenching rate of the excited ZnPP triplet state within the hemoglobin by oxygen, methyl viologen, and anthraquinonesulfonate served as a measure of the migration rate through the protein into the binding site. It was found that the activation energies for all three quenchers were very similar and closely resembled those in myoglobin, suggesting that the migration rates are determined by the subunit structure only and that the quaternary configurational changes do not influence the quenching rates. The implications of the results for electron transfer in proteins are briefly discussed.     

Inhibition of Pyrimidine Dimer Formation in DNA by Cationic Molecules: Role of Energy Transfer

In addition to the acridine dyes, acridine orange and proflavine, we find that three other cationic molecules which bind to DNA-ethidium bromide, chloroquine, and methyl green-inhibit the production of cyclobutyl pyrimidine dimers by ultraviolet radiation. Intercalation is not necessary for dimer inhibition. The long range nature of the inhibition implies that energy transfer is responsible. The transfer is between the lowest excited singlet state of DNA and the acceptor singlet, and seems to involve the F?rster mechanism.     

Photophysics of tryptophan in bacteriophage T4 lysozymes

Bacteriophage T4 lysozyme contains three tryptophan residues in distinct environments. Lysozymes with one or two of these residues replaced by tyrosine are used to characterize the photophysics of tryptophan in these individual sites. The fluorescence spectra, average lifetimes, and quantum yields of these three single-tryptophan variants are understandable in terms of the neighboring residues. The emission spectra and radiative lifetimes are found to be the same for all three species while the quantum yield and decay kinetics are quite distinct. The variation of the average nonradiative rate constant is correlated with neighboring quenching groups. Quenching by I- correlates with exposure of the tryptophan residue based on the crystal structure. Complex behavior is observed for the time dependence of the fluorescence decay in all three cases, including that of the immobile tryptophan-138 residue. The complexity of the fluorescence decay is ascribed to heterogeneity in the nonradiative rate constant among microstates. Energy transfer between tryptophan residues is inferred to occur from comparison of the quantum yields of the two-tryptophan and single-tryptophan proteins and is discussed in terms of the F?rster mechanism.     

Kinetics of intramolecular contact formation in a denatured protein

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule F?rster resonance energy transfer (FRET) experiments.     

Detection of Tryptophan to Tryptophan Energy Transfer in Proteins

Förster resonance energy transfer (FRET) studies usually involve observation of intensity or life-time changes in the donor or acceptor molecule and usually these donor and acceptor molecules differ (heterotransfer). The use of polarization to monitor FRET is far less common, although it was one of the first methods utilized. In 1960, Weber demonstrated that homotransfer between tryptophan molecules contributes to depolarization. He also discovered that the efficiency of homotransfer becomes much less effective upon excitation near the red-edge of the absorption. This “red-edge effect” was shown to be a general phenomenon of homotransfer. We have utilized Weber's red-edge effect to study tryptophan homotransfer in proteins. Specifically, we determined the polarization of the tryptophan fluorescence upon excitation at 295 nm and 310 nm (near the red-edge). Rotational diffusion leads to depolarization of the emission excited at either 295 nm or 310 nm, but homotransfer only contributes to depolarization upon excitation at 295 nm. Hence, the 310/295 polarization ratio gives an indication of tryptophan to tryptophan energy transfer. In single tryptophan systems, the 310/295 ratios are generally below 2 whereas in multi-tryptophan systems, the 310/295 ratios can be greater than 3.     

Förster Energy Transfer in the Vicinity of Two Metallic Nanospheres (Dimer)

We present a detailed theoretical analysis of the Förster energy transfer process when a pair of molecules (donor and acceptor) is located nearby a cluster of two metallic nanospheres (dimer). We consider the case in which plasmonic resonances are within the overlap between the donor emission and acceptor absorption spectra, as well as the case that excludes such resonances from the aforementioned spectral overlap. Moreover, we explore the dependence of the Förster energy transfer rate on different dimer configurations (size and separation of nanospheres) and several dipole orientations of molecules. The dimer perturbs strongly the Förster energy transfer rate when plasmons are excited, donor dipole is oriented along the longitudinal axis of the dimer, and the radii of nanospheres and the sphere-gap distance are on the order of a few nanometers. In case of plasmonic excitation, the Förster energy transfer rate is degraded as the sphere-gap distance and size of the nanoparticles increase due to the dephasing of electronic motion arising from ohmic losses of metal. Also, we study the Förster efficiency influenced by the dimer, finding that the high efficiency region (delimited by the Förster radius curve) is reduced as a consequence of significant enhancement of the direct donor decay rate. Our study could impact applications that involve Förster energy transfer.     

Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer

A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to F?rster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The F?rster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.     

Photochemically induced electron transfer

Biochemical reactions involving electron transfer between substrates or enzyme cofactors are both common and physiologically important; they have been studied by means of a variety of techniques. In this paper we review the application of photochemical methods to the study of intramolecular electron transfer in hemoproteins, thus selecting a small, well-defined sector of this otherwise enormous field. Photoexcitation of the heme populates short-lived excited states which decay by thermal conversion and do not usually transfer electrons, even when a suitable electron acceptor is readily available, e.g., in the form of a second oxidized heme group in the same protein; because of this, the experimental setup demands some manipulation of the hemoprotein. In this paper we review three approaches that have been studied in detail: (i) the covalent conjugation to the protein moiety of an organic ruthenium complex, which serves as the photoexcitable electron donor (in this case the heme acts as the electron acceptor); (ii) the replacement of the heme group with a phosphorescent metal-substituted porphyrin, which on photoexcitation populates long-lived excited states, capable of acting as electron donors (clearly the protein must contain some other cofactor acting as the electron acceptor, most often a second heme group in the oxidized state); (iii) the combination of the reduced heme with CO (the photochemical breakdown of the iron-CO bond yields transiently the ground-state reduced heme which is able to transfer one electron (or a fraction of it) to an oxidized electron acceptor in the protein; this method uses a "mixed-valence hybrid" state of the redox active hemoprotein and has the great advantage of populating on photoexcitation an electron donor at physiological redox potential).     

Reactivities of tyrosine, histidine, tryptophan, and methionine in radical pair formation in flavin triplet induced protein nuclear magnetic polarization

In order to check the validity of several basic assumptions of protein photochemically induced nuclear polarization (protein photo-CIDNP), we have investigated the quenching processes of the dye triplets by the side chains of tyrosine, histidine, and tryptophan in a variety of molecular systems and environments. The quenching (H atom or electron transfer) is the generating process of the triplet electron-spin-correlated radical pair, the evolution of which gives rise to nuclear polarization. At pH 7 the quenching of 10-(carboxyethyl)flavin triplets by tyrosine and tryptophan is almost diffusion controlled. Quenching by histidine is slower. We have also investigated the slow quenching (by electron transfer) by the side chains of methionine and could show that quenching by cysteine S derivatives is negligible. Quenching by tyrosine and histidine peptides and by the tyrosines of the pancreatic trypsin inhibitor protein is slightly slower than by free side chains. Quenching is strongly viscosity controlled, to be expected of a process requiring bimolecular contact. Reactivity trends at high viscosities resemble those observed in fluid aqueous solutions. Activation energies of quenching by tyrosine, tryptophan, and histidine are similar. No difference could be detected in the mechanism of quenching by these side chains. No fast static quenching was observed that could compete with the diffusional process.     

Triplet-singlet energy transfer in the complex of auramine O with horse liver alcohol dehydrogenase

Triplet-singlet energy transfer has been studied in the complex formed between auramine O (AO) and horse liver alcohol dehydrogenase with optically detected magnetic resonance (ODMR) spectroscopy. The results show that Trp-15 and Tyr residues transfer triplet energy mainly by a trivial process, whereas Trp-314 transfers triplet energy by a F?rster process with two observed lifetimes at 77 K of 170 and 50 ms. The different F?rster energy-transfer lifetimes are ascribed either to quenching of the two Trp-314 residues of the dimer by a single asymmetrically bound AO or to two distinct conformations of the enzyme-dye complex with differing separations and/or orientations of donor and acceptor. Individual spin sublevel transfer rate constants are reported for the major decay component with the 170-ms Trp triplet-state lifetime; these are found to be highly selective with kxtr much greater than kytr and kztr.     

Quenching of room temperature protein phosphorescence by added small molecules

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of oxygen on the amplitude of photodriven electron transfer across the lipid bilayer-water interface.

The surprisingly small effect of oxygen on photoelectron transfer in pigmented lipid bilayers is traced to a short lifetime of the excited states. Decreasing the oxygen concentration by greater than 100-fold decreases the half saturating concentration of acceptor by only threefold and has no effect on the maximum photovoltage observed at acceptor saturation. This holds true for both magnesium octaethylporphyrin and chlorophyll with both ferricyanide and methyl viologen as acceptors. Since oxygen quenches excited states at near the encounter limit, the lifetime of reactive state must be short, less than 100 ns. About 100-fold higher concentrations of acceptor are required to quench the fluorescence (in liposomes) than to saturate the photoeffect. Thus the reactive state is most likely the triplet. The short life of the excited state is caused by concentration quenching, i.e., their reaction with ground state molecules. The increase of photovoltage with increasing pigment concentration shows that this quenching in a condensed form of the pigment produces ions that lead to the observed photovoltage by interfacial reaction of the anion with acceptor.     

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.     

Quenching of excited states of chlorophyll molecules in submembrane fractions of Photosystem I by exogenous quinones

The ability of three substituted quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duriquinone) to quench the excited states of chlorophyll (Chl) molecules in Photosystem I (PSI) was studied. Chl fluorescence emission measured with isolated PSI submembrane fractions was reduced following the addition of exogenous quinones. This quenching progressively increased with rising concentrations of the exogenous quinones according to the Stern-Volmer law. The values of Stern-Volmer quenching coefficients were found to be 3.28 x 10(5) M(-1) (DBMIB), 1.31 x 10(4) M(-1) (DCBQ), and 3.7 x 10(3) M(-1) (duroquinone). The relative quenching capacities of the various exogenous quinones in PSI thus strictly coincided to those found for the quenching of Fo level of Chl fluorescence in isolated thylakoids, which is emitted largely by Photosystem II (PSII) [Biochim. Biophys. Acta (2003) 1604, 115-123]. Quenching of Chl excited states in PSI submembrane fractions by exogenous quinones slowed down the rate of P700, primary electron donor of PSI, photooxidation measured at limiting actinic light irradiances thus revealing a reduced photochemical capacity of absorbed quanta. The possible involvement of non-photochemical quenching of excited Chl states by oxidized phylloquinones, electron acceptors of PSI, and oxidized plastoquinones, mobile electron carriers between PSII and the cytochrome b(6)/f complex, into the control of photochemical activity of PSI is discussed.     

An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans

Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.     

Penetration of an emulsion surface by cholesteryl ester transfer protein

Quenching of the intrinsic fluorescence of cholesteryl ester transfer protein (CETP) by spin labelled fatty acids (5-NS and 16-NS) was investigated to determine the degree to which the protein penetrated the phospholipid monolayer surface of a lipid emulsion. When bound to the phospholipid surface approximately 50% of the fluorophores of the transfer protein were accessible to quenching by 5-NS whose nitroxy group locates near the monolayer surface. On the other hand, only 22% of the fluorophores of CETP were accessible to quenching by 16-NS whose nitroxy group locates deeper in the surface monolayer. Quenching of the CETP fluorescence by an aqueous phase quencher (acrylamide) shows that the protein undergoes a conformational change on binding which increases the proportion of the tryptophan residues exposed to the aqueous phase. The results indicate that CETP does not penetrate the lipid surface to a significant degree. Received: 29 March 1996 / Accepted: 30 May 1996     

Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Proton magnetic resonance characterization of the dynamic stability of the heme pocket in myoglobin by the exchange behavior of the labile proton of the proximal histidyl imidazole.

The assigned exchangeable proton signals in the proton nuclear magnetic resonance spectra of sperm whale deoxy and Met-cyano myoglobin in H2O solution were found to exhibit pH-dependent saturation transfer from the bulk water, which allowed determination of the kinetics and mechanism of the labile proton exchange with solvent. The exchange rates are base catalyzed for both protein forms, with the rate eight times faster in Met-cyano than in deoxy myoglobin. The exchange rate is taken as a measure of the magnitude of the fluctuation in the protein conformation near the heme cavity. On the basis of tritium exchange methods, the greater stability of the unligated relative to the ligated state in myoglobin has also been reported for hemoglobin. The present study, however, localizes the differential kinetic stability on the F helix whose flexibility has been implicated in the mechanism of cooperativity. The observation that filling the hydrophobic vacancy on the proximal side of the heme near the proximal histidine in Met-cyano myoglobin wih cyclopropane increases the proton lability argues against the role for this hole in facilitating the flexibility of the F helix in the native protein.     

pH-dependence of photo-induced electron transfer in zinc-substituted sperm whale myoglobin

The electron transfer between the excited triplet state of zinc-substituted sperm whale myoglobin and Cu2+ has been studied by following the decay rate of delayed fluorescence. The Cu2+ bound on the surface of the myoglobin molecule are efficient quenchers of the excited electron state of Zn-myoglobin. Two bimolecular rate constants of quenching (KQ) for every pH investigated have been calculated. The pH-dependence of KQ1 indicates that the protonation of one amino acid residue (His-GH1 (119] is important for the process. Our results support the idea of the common nature of the mechanism of quenching by Cu2+ and oxidation of oxymyoglobin by Cu2+.