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.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime and anisotropy studies with liver alcohol dehydrogenase and its complexes

From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.     

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.     

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Conformation of coenzyme fragments when bound to lactate dehydrogenase

The conformations of adenosine, 5′-AMP and 5′-ADP when bound to dogfish M₄ lactate dehydrogenase at pH 7.8 or greater have been determined at 2.8 Å resolution to investigate the events on coenzyme binding. The coenzyme fragments AMP and ADP induce a conformational change in lactate dehydrogenase at pH values less than 6.0 in the same way as do NAD⁺, NADH or ADPR at any pH value. The structure of NAD⁺ when bound to lactate dehydrogenase had previously been determined at 5.0 Å resolution. The structures of the bound adenosine, AMP, ADP and NAD⁺ are compared with the preliminary structure of NAD in a 3.0 Å resolution map of the ternary complex LDH-NAD—pyruvate. Small but significant changes in the binding of the phosphates could be important in the folding of the protein loop over the substrate binding pocket.     

Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes.

Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.     

Luminescence study of the conformation behavior of alcohol dehydrogenase from horse liver during substrate binding

The luminescence quenching and conformational behavior of alcohol dehydrogenase from horse liver upon substrate binding has been studied. It was shown that the binding of NADH and NAD+ to the enzyme resulted in the quenching of Trp-314 luminescence, whereas the luminescence of Trp-15 was not quenched. In this case non-radiating energy transfer from Trp-314 to NADH was observed. An essential energy transfer from Trp-15 to NADH and between the two Trp-314 of both subunits of the enzyme was not revealed. The quenching of the enzyme luminescence upon NAD+ binding was, mainly, caused by NAD+ reduction up to NADH. It was assumed, that the release of the proton upon NAD+ binding occurred due to the reduction. Binding of ethanol, ADP or adenosine did not result in essential conformational changes of the enzyme.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

Circular dichroism of complexes of NADH with self-associating bovine liver glutamate dehydrogenase.

The CD of GDH–NADH complexes was measured in order to reexamine the binding of coenzyme to GDH. The existence of two distinct Cotton effects associated with two separate NADH binding sites/subunit was confirmed with native, polymerizing and crosslinked, unpolymerizing enzyme. CD titration of the high-affinity NADH sites revealed significant dependence of the optical activity of the bound coenzyme on the state of protein association. Molar ellipticity of bound NADH decreased with the increasing degree of polymerization of GDH. It is suggested that the high-affinity NADH sites are loacted at or near the association interface. Binding of NADH to the low-affinity sites, in the presence of GTP, leads to an inversion of the CD spectrum of GDH–NADH complexes. This inversion is not related to the polymerization of GDH. However, for proper analysis of the CD of NADH bound to the low-affinity sites, a correction for the effect of polymerization on the optical activity of NADH bound to the high-affinity sites is required.     

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.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Pulse fluorimetry study of octopine dehydrogenase-reduced nicotinamide adenine dinucleotide complexes.

We measured the transient fluorescence of NADH bound to octopine dehydrogenase in the binary octopine dehydrogenase-NADH complex and in the ternary complexes containing D-octopine, L-allooctopine, L-arginine, D-arginine, or 5-guanidinovaleric acid. The fluorescence decay in all these complexes is biexponential. This is explained by the presence of several conformations of the single NADH binding site. In addition, transietn anisotropy measurements show that the nicotinamide moiety is rigidly bound to the enzyme.     

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.     

Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core

The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.     

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.     

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.     

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.