Partial Quenching of Parametrical Tetragonality of a Chromium(III) Complex by Pressure Tuning.

The ligand-field spectrum of [Cr(NH(3))(4)F(2)]AsF(6) has been pressure-tuned up to 100 kbar. The parametrical analysis shows that the absolute values of the two independent tetragonalities of the chromophoric ion both decrease with increasing isotropic pressure so that the molecular ion becomes more isotropic. Moreover, the Cr-N single bonds appear to be more compressible than the Cr-F bonds, for which it is parametrically clear that they have a partial multiple-bond character. While the spectrochemical parameter for F(-), Delta(F), increases with pressure, its two components Delta(sigma)(F) and Delta(pi)(F) decrease. It is confirmed that nephelauxetism increases with pressure.

Effect of hydrostatic pressure on lysozyme and chymotrypsinogen detected by fluorescence polarization.

The effect of hydrostatic pressure upon solutions of chymotrypsinogen and lysozyme at room temperature has been followed by employing a new technique [Chryssomallis, G. S., Drickamer, H. G., & Weber, G. (1978) J. Appl. Phys. 49, 3084] that permits the measurement of fluorescence polarization at pressures of up to 10 kbar. Lysozyme shows a stable, reversible 60% increase in apparent volume when the pressure is raised to 9 kbar. This can be given a simple interpretation in terms of solvent penetration of the structure at higher pressures. In contrast, the results with chymotrypsinogen are time dependent and only partially reversible on release of the pressure. They involve conversion (tl/e = 5 min) to a form with a lower rotational rate at approximately 6 kbar and return to a fast-rotating form at higher pressure. This latter form persists on pressure release. The possibility of generating what are clearly metastable conformations, not only in chymotrypsinogen but also in flavodoxins [Visser, A. J. W. G., Li, T. M., Drickamer, H. G., & Weber, G. (1977) Biochemistry 16, 4879], indicates that there are unresolved questions about the relative stability of protein conformations which can be profitably investigated by high-pressure experiments.

Effect of hydrostatic pressure upon ethidium bromide association with transfer ribonucleic acid.

The binding of ethidium bromide to yeast phenylalanine-specific transfer ribonucleic acid (tRNAPhe) has been investigated in the pressure range from 1 atm to 9 kbar in the presence of 100 mM sodium chloride and 10 mM magnesium chloride, pH 7.7. One high-affinity binding site for ethidium is present, with a dissociation constant of 2.4 X 10(-6) M at 1 atm and 22 degrees C. Binding to this site is enhanced with increasing pressure, the dissociation constant reaching 2.9 X 10(-7) M at 2 kbar. Pressure also promotes the binding of ethidium to lower affinity sites of tRNAPhe. The standard volume change upon complex formation is found to be 25.6 +/- mL/mol for the first ethidium bound. If sodium is replaced by lithium in the buffer, the standard volume change is 23.3 +/- 0.5 mL/mol. We conclude that decrease of the electrostatic repulsion in the negatively charged tRNAPhe by binding of the positively charged ethidium is the main cause of the relatively large volume decrease upon complex formation. The electrostatic repulsion that must be present in this case, as well as in other nucleic acids, implies that intercalating binding sites are of the "soft" type as previously defined [Torgerson, P.M., Drickamer, H.G., & Weber, G. (1979) Biochemistry 18, 3079]. Model studies by others of the binding site characteristics are in agreement with this concept.

Volume changes in the formation of internal complexes of flavinyltryptophan peptides.

The effect of pressure, up to 10 kbar, on the fluorescence yield and lifetime of two flavinyltryptophan peptides was investigated. These peptides differed only in the number of methylene groups, respectively three and five, separating the chromophores. At atmospheric pressure the closed nonfluorescent form predominated in both compounds constitutin 94% of the total in the short-linked peptide and 80% in the long-linked one. The fluorescence of both peptides decreased at high pressure and the volume change upon formation of the nonfluorescent complex in the short peptide (--1.8 mL/mol) was less than half of the change in the long peptide (--4.8 mL/mol) or the value for FAD (--4.3 mL/mol). The much smaller compressibility of the short peptides is attributed to the mechanical constraint to the approach of the interacting rings, imposed by the short link. Mechanical constraints of similar nature may be expected to be operative in proteins. Their importance in pressure denaturation is discussed.

Effect of pressure upon the fluorescence of various flavodoxins.

The effects of hydrostatic pressure in the range of 10(-3) to 11 kbar on the fluorescence of flavodoxins from Peptostreptococcus elsdenii, Desulfovibrio vulgaris, Azotobacter vinelandii, and Clostridium MP were investigated. The first three flavoproteins showed under high pressure enhancements of flavin fluorescence of over 50 times resulting from the release of flavin mononucleotide from the protein complex. The Clostridial flavodoxin showed a very much smaller fluorescence change. At pH 7.5 the high-pressure fluorescence changes of the flavodoxins of D. vulgaris and P. elsdenii were not reversed by decompression, but in A. Vinelandii the pressure changes were over 80% reversible. At pH 5 over 80% reversibility was restored to the flavodoxins of D. vulgaris and P. elsdenii, although the pressure dependence of the fluorescence changes was very similar in the reversible and irreversible cases. The midpoint pressures in the reversible reactions were 4.7 kbar (D. vulgaris), 8.7 kbar (P. elsdenii), and 10.6 kbar (A. vinelandii) indicating specific differences in the flavin binding regions. Apparent volume changes in these reactions were 65-75 mL/mol indicating participation of a large fraction of the protein in the pressure-induced changes. The irreversible changes are not related to protein aggregation and are believed to result from a pressure-dependent covalent modification, not yet characterized, of the flavin binding region of the protein.

Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex.

The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5 kbars and the change in volume, delta, in the reaction (native-to-denatured) was calculated to be -74ml/mol. Addition of up to 30% methanol results in a progressive decrease both in delta and rho 1/2, in agreement with the concept that hydrophobic bonding stabilizes the native structure.

High pressure studies of solvent effects on anthracene spectra.

Measurements have been made of the effect of pressure on the peak location and peak shape for anthracene in a variety of liquid and plastic environments; both absorption and fluorescence studies were made. The results are discussed from two standpoints: in terms of the dielectric model and of a configuration coordinate model. For the latter model, the change of configuration coordinate (volume decrease of the system upon electronic excitation) is shown to correlate well with the product of the compressibility and polarizability of the solvent. For the dielectric model, it is found that the change of cavity volume with density is complex. However, the relative cavity volume obtained from emission measurements was consistently 10-15% smaller than that obtained from absorption. The cavity volume decreased with increasing polarizability of the solvent, and results obtained from absorption and emission were quite consistent in this regard.

The evaluation of configuration coordinate parameters from high pressure absorption and luminescence data.

A configuration coordinate model was previously developed for relating the energies involved in optical and thermal electron excitations between two states and the effect of pressure on these energies. In the course of this development, expressions were presented for hv(max), the optical absorption peak maximum, and (E((1/2)))(a) the peak half-width, as functions of pressure, and of the parameters defined in the model. The analysis was later extended to emission. We elaborate on the equations for the change of hv(max) and E((1/2)) with pressure and discuss some of the problems involved in evaluating and using peak half-widths. Various combinations of absorption and emission data for evaluating the parameters describing the excitation are discussed and are compared for phenanthrene in various environments.

The effect of pressure on the molecular complex of isoalloxazine and adenine.

The effect of pressure to 10 kilobars on the fluorescence characteristics of flavin mononucleotide (FMN), flavin-adenine dinucleotide (FAD), and on complexes of FMN with adenylic acid (AMP), and with I(-) has been studied. The properties measured include peak location, fluorescence yield, and lifetime. From these results the equilibrium constant K and the rate constant k(+) (*) for complex formation were evaluated as a function of pressure. The pressure dependence of these coefficients shows that the volume of the system decreases upon complex formation and that there is an expansion upon formation of the activated complex (DeltaVdouble dagger is positive). The implications of these results for protein denaturation are mentioned.

High-pressure spectral studies of mixed valence compounds of antimony.

In Cs(2)SbCl(6) and related compounds antimony appears as Sb(III) and Sb(V) in alternate halide octahedra. The optical spectrum contains "mixed valence" peaks assigned to Sb(III) --> Sb(V) transfer near 18 and 27 kK (cm(-1)). In addition there is a peak near 31 kK assigned to an internal transition on Sb(III) and one near 37 kK assigned to Sb(V), mixed with the absorption edge of the crystal. The mixed valence peaks shift strongly to lower energy with pressure (about 5 kK in 120 kbar), and decrease rapidly in integrated intensity, as does the Sb(III) peak near 31 kK. A new peak appears near 33-34 kK, tentatively assigned to Sb(IV). The ground state apparently transforms from Sb(III)-Sb(V) to Sb(IV)-Sb(IV) at high pressure. Similar behavior is observed for Cs(2)Sb(0.3)Sn(0.7)Cl(6) and (CH(3)CH(2)NH(3))(2)Sb(0.5)Sn(0.5)Cl(6).

Optical versus thermal transitions in solids at high pressure.

Pressure-induced electronic transitions have been observed in a wide variety of materials. In particular, Mössbauer resonance studies under high pressure have revealed changes of oxidation state and spin state of iron as a function of pressure. These processes involve the thermal transfer of an electron to a new ground state of the system. The difference in energy between states is frequently measured by optical absorption. In this paper, we relate the energy of the optical absorption peak and its half-width to the difference in thermal energy between the two states as a function of pressure. We show that the optical peak will broaden with pressure only if there is a difference between the force constants of the ground and excited states. These relationships permit the prediction from optical absorption data of the pressure at which a new ground state will be obtained. We demonstrate that the predictions are qualitatively correct for the reduction of Fe(III) in a series of ferric hydroxamates, and for the low-spin to high-spin transition of Fe(II) in ferrous phenanthroline complexes.

Effect of pressure on the electronic structure of ferric hydroxamates and ferrichrome A.

The effect of pressure up to 175 kilobars on the electronic structure of three ferric hydroxamates and on ferrichrome A has been studied by optical absorption and Mössbauer resonance. The ferric ion was reduced to ferrous ion with pressure, as has been previously observed for various compounds. For the hydroxamates, the amount of reduction correlated very well with the location and shift of the metal-to-ligand charge transfer peak. This is entirely consistent with a previously presented theory. The results for ferrichrome A did not fit quantitatively into the series. Since the shape of the potential well is almost certainly different for this compound, this result is not surprising.

The effect of pressure on the oxidation state of iron, iv. Thiocyanate and isothiocyanate ligands.

The effect of pressure on the Mössbauer resonance spectra of Fe(III) with thiocyanate (M-SCN) and isothiocyanate (M-NCS) ligands has been studied. Fe(NCS)(3).6H(2)O, which has the isothiocyanate structure, reduces with increasing pressure, reversibly, and with a pressure dependence for the conversion very similar to that shown by a wide variety of ionic ferric compounds. K(3)Fe(SCN)(6) has the thiocyanate structure. At low pressures, it exhibits a significantly larger reduction than the Fe(NCS)(3). With increasing pressure the thiocyanate complexes isomerize, each complex apparently exhibiting about the same degree of conversion at a given pressure. At 150 kb the isomerization is essentially complete. The reduction of the Fe(III) to Fe(II) is reversible but the isomerization is not, and the sample, when powdered and reloaded in the high-pressure cell, exhibits the isomer shift, quadrupole splitting, and Fe(III) to Fe(II) conversion characteristic of an isothiocyanate. Heating the thiocyanate to 110 degrees C at 5 kb yields a mixture of thiocyanate and isothiocyanate that converts with pressure completely to the isothiocyanate.