Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

USE OF TRIS (2,2'-BIPYRIDINE) RUTHENIUM(II) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

Abstract— A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H₂O and D₂O. The technique uses tris (2,2'-bipyridine) ruthenium(II) dication (Ru(bipy)₃₂₊) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of Ru(bipy)₃2+* can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (±0.3) x 10⁶ mol^-1 dm³ s^-1 has been determined for the reaction of ¹O₂ with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (±0.4) x 10⁶ and 3.1 (±2.0) x 10⁵ mol^-1 dm³ s^-1; for swine skin gelatin this value is 3.9 (±2.4) × 10⁵ mol^-1 dm³ s^-1. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and ¹O₂.     

Interaction between Cytochrome b2 Core and Flavodehydrogenase from the Yeast Hansenula anomala

Abstract— The binding of cytochrome b₂ core (a monomer) to flavodehydrogenase (a tetramer), both purified from Hansenula anomala flavocytochrome b₂, has been studied in the presence of 2- p -toluidinylnaphthalene-6-sulfonate (TNS). The association constant of the TNS-flavodehy-drogenase complex was found to be equal to 0.64 μ M ⁻¹ with a stoichiometry of one TNS per tetramer. Binding of cytochrome b₂ core to flavodehydrogenase was followed by monitoring changes in the TNS fluorescence. Our results indicated that the binding is cooperative, with a stoichiometry of four cytochrome b₂ cores per tetramer of flavodehydrogenase.     

QUENCHING OF EXCITED TRIPLET LUMIFLAVIN BY LUMIFLAVIN RADICALS FORMED IN ITS T-T REACTION

Abstract— Reactions of the triplet state of lumiflavin (³LF) in water adjusted at pH 7.2 were reexamined by means of a Xe-flash photolysis and a laser photolysis. Measurements of the decay of ³LF were made on solutions of LF ranging in the concentration from 4 to 61 times 10^-6 mol/dm³. A one-electron reduced and a one-electron oxidized species of lumiflavin (LF^- and LF⁺) were produced in the first decay stage of ³LF with a high efficiency (0.6 ± 0.1) in a bimolecular triplet-triplet reaction. The product radicals (LFH^- and LF⁺) quench ³LF very efficiently (3 ± 0.8 × 10⁹ mol^-1dm³ s^-1) compared with LF in the ground state (> 2 × 10⁷ dm³ mol^-1).     

THE PHOTOCHEMICAL INTERACTION BETWEEN THE TRIPLET STATE OF 8-METHOXYPSORALEN AND THE MELANIN PRECURSORL–3, 4 DIHYDROXYPHENYLALANINE

Abstract— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH₂) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH₂ ( k ∼ 2 × 10⁹ dm³ mol^-1 s^-1) to form semireduced 8-MOP and semioxidised dopaH₂.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH₂ at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λ_max and extinction coefficients found were: for 8-MOP at pH 6.5, λ_max= 350 nm (= 9050 dm³ mol^-1 cm^-1); for dopa at pH 6.5, λ_max= 305 nm (ε= 12000 dm³ mol^-1 cm^-1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm³ mol^-1 cm^-1).     

THE CHARACTERISATION OF THE TRIPLET STATE OF CROCETIN, A WATER SOLUBLE CAROTENOID, BY NANOSECOND LASER FLASH PHOTOLYSES

Abstract— The triplet state of crocetin, which is a water soluble carotenoid, has been sensitized by psoralen. The triplet extinction coefficient, ε_T (73000 dm³ mol^-1 cm^-1 at 470 nm), the triplet-triplet spectrum and the quantum yield of triplet formation, φ_T (less than 1%) are reported in aqueous solution.
In order to calculate the extinction coefficient of crocetin it was necessary to obtain ε_T for psoralen in water (10000dm³ mol^-1 cm^-1 at 450 nm). This latter value was obtained using the complete conversion technique and is reported with the triplet-triplet spectrum.     

Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibiotics can induce skin photosensitivity in some patients and this has been ascribed to the generation of reactive oxygen species, such as singlet oxygen (O₂[¹Δ_g]). We have studied the photochemical properties of the different ionized forms of the fluoroquinolone norfloxacin upon complexation with Mg²+ and Ca²+ ions, as it is proposed that the antibiotic exists mainly as a complex in the blood plasma. We found that the norfloxacin cation (pH < 6) shows no photodegradation after UVA irradiation and has a low quantum yield of O₂(¹Δ_g) generation. The norfloxacin cation does not complex. Ca²+ or Mg²+ ions; when these ions are added to the solution, we observed no changes in the fluorescence quantum yields (φ_flu) and singlet oxygen yields (φ_Δ). In contrast, the neutral (6 ± pH > 8.5) and anionic (pH > 9) forms of norfloxacin are able to complex calcium and magnesium, and their generation of O₂ (¹Δ_g) is decreased by complexation. The neutral zwitterionic form and the anionic form also quench singlet oxygen by both chemical and physical pathways regardless of complex formation, while physical quenching is observed for the cation. At pH > 7.4, norfloxacin photobleaches and complexation to Ca²+ and Mg²+ increases the rate at which photobleaching occurs. Thus, both the pH of the medium and complexation with metal cations may affect the phototoxic potential of this antibiotic.     

THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS

Abstract— The physical quenching of singlet molecular oxygen (¹Δ_g) by amino acids and proteins in D₂O solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1–06 μm output of a Nd-YAG laser, which absorbs directly in the electronic transition ¹Δ_g+ 1 v →³Σ_g^-. The rate of quenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-blue-photosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in D₂O, pD 8, are (10⁷ℒ mol^-1 s^-1): alanine 0–2, methionine 3, tryptophan 9, histidine 17, carbonic anhydrase 85, lysozyme 150, superoxide dismutase 260, aposuperoxide dismutase 250.     

SPECTROSCOPIC EXAMINATION OF HYDROGEN BONDING and PROTON TRANSFER IN MODEL SCHIFF BASES RELATED TO THE VISUAL PIGMENT CHROMOPHORE

By means of a variable low temperature cell holder, double beam spectra were obtained to temperatures as low as 88 K. As the temperature was lowered for a 3McP solution containing N-2,4,6,8,10-dodecapentaenylidene-n-butylamine (2) and phenol, three separate species could be detected, (i) The first spectrum taken at room temperature exhibits sharp fine structure and is the unperturbed spectrum of 2. As the temperature is lowered, a completely new spectrum with fine structure develops at slightly longer wavelength while simultaneously the old fine structure disappears. (ii) The new spectrum belongs to the H-bonded species formed between 2 and phenol. As temperature is further lowered, the second spectrum disappears as a new band develops at about 440 nm. (iii) This is the proton transferred species. The same phenomenon is repeated when p-nitrophenol and acetic acid are substituted for phenol. The -ΔH and ΔS values for the H-bonding process between phenol and 2 in 3MeP are 3900 cal mol^-1 and 1.4 cal dgr^-1 mol^-1 respectively. Additional experiments firmly establish the process to be H-bonding with the phenolic hydroxyl group. Alcohol also H-bonds with 2 but does not proton transfer.     

THE EFFECT OF MOLECULAR POLARIZATION ON THE ELECTROCHROMISM OF CAROTENOIDS—I. THE INFLUENCE OF A CARBOXYLIC GROUP

Abstract— Electrochromism of oriented all- trans -β-apo-8'-carotenoic acid is studied in thin capacitors. The linear electrochromism is very strong, in contrast to that of symmetrical carotenoids. It is proportional to the first derivative of the absorption spectrum. The quadratic electrochromism can be described as a superposition of fractions proportional to the first and second derivatives of the absorption spectrum. The permanent dipole moment difference between the ground state and the excited state of the carotenoic acid molecule is Δμ= 3.6 × 10^-29 C·m (±20%) (10.7 Debyes). The polarizability difference parallel to the long axis of the molecule is Δα|| = 1.17 × 10^-37 C·m²·V^-1 (±20%) (1050 ų). Furthermore, the relative permittivity of the solid carotenoic ethyl ester is _r= 3.5 ± 0.2.
Δμ is due to the polarizing force of the carboxylic group. This force is equivalent to a mean local electric field of F _t≅3 × 10⁶V/cm. Such a "local field" may also be exerted on a symmetrical carotenoid in the membrane of photosynthesis, e.g. by asymmetrical complex formation with a polarizing molecule. To obtain an effective permanent field of F _p≅ 2 × 10⁶V/cm across the membrane, as postulated in photosynthesis, a local field of F _l≅ 5.5 × 10⁵ V/cm would be sufficient. F _p is shown to be directed from inside to outside of the thylakoid. To realize this, e.g. a positive polar (i.e. electron-attracting) complex partner of the carotenoid, located more to the inside of the thylakoid, can be postulated.     

INHIBITION OF ETHYL BENZENE OXIDATION BY STABLE IMINOXY RADICALS, STUDIED BY CHEMILUMINESCENCE

Abstract— The inhibition by stable radicals having the structure (X-O)₂NO of the initiated oxidation of ethyl benzene was studied. According to a formal kinetic scheme, verified by both theoretical computer calculations and an experimental chemiluminescence method, the rate constants of the interaction of peroxyradicals with iminoxy radicals were estimated at 60°C to be: 2.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₃ and 3.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₂CH₃.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

FLUORESCENCE QUENCHING OF INDOLIC COMPOUNDS BY ALIPHATIC AMINO ACIDS. EVIDENCE FOR EXCITED STATE COMPLEXES

Abstract The fluorescence quenching of indole, tryptophan, tryptamine and indole-3-acetic by aliphatic amino acids was studied. The bimolecular rate constant ( k _q) for the deactivation of the excited state was determined. The k _q values were in the range 0.6 × 10⁸–1.6 × 10⁹ M ^–1 S^–1 and they increased in the order tryptophan < tryptamine < indole ≈ indole-3-acetic acid. When the rate constant was corrected for diffusion al effects a good linear correlation was found between the log ( k '_q) and the ionization equilibrium constant of the carboxylic group of the amino acid (p ^k _a1). This was interpreted as arising from a charge transfer mechanism in which the indole moiety acts as an electron donor and the carbonyl group of the amino acid as the acceptor.
The activation parameter for the quenching processes were also determined. The ΔH^≠ values were in the range —4.0 to +4.0 kcal/mol and the ΔH^≠ in the range –7 to –37 e.u. For the systems with lower values of k _q negative values for ΔH^≠ were observed. A good enthalpy-entropy compensation was found with an isokinetic temperature of 229 K. These results suggest that a common mechanism is operating for all the systems and that it involves the formation of an excited state complex between the indolic compound and the amino acid.     

SINGLET OXYGEN YIELDS AND RADICAL CONTRIBUTIONS IN THE DYE-SENSITISED PHOTO-OXIDATION IN METHANOL OF ESTERS OF POLYUNSATURATED FATTY ACIDS (OLEIC, LINOLEIC, LINOLENIC AND ARACHIDONIC)

Abstract— The triplet state characteristics (spectrum, lifetime and quantum yield) for four dye sensi tisers [methylene blue (MB), erythrosin (ER), haematoporphyrin (HP) and riboflavin (RF)] were determined in methanol by laser flash photolysis and singlet oxygen yields (0.60 to 0.48) from time-resolved measurements of the 1270 nm near infrared emission. The reaction of singlet oxygen with four long chain unsaturated phenyl esters [oleate (18: 1), linoleate (18: 2), linolenate (18: 3) and arachidonate (20: 4)] was followed quantitatively using the singlet oxygen luminescence technique and also, after continuous420–700 nm irradiation, by HPLC and other analysis of the isomeric product monohydroperoxides. The overall quantum yield of photooxidation (∼10^-2) was shown to be consistent with the observed singlet oxygen quenching constants(2–12 times 10⁴ dm³ mol^-1 s^-1) for the four esters studied and the singlet oxygen lifetime in methanol (τ∼ 9 μs). The isomer product distribution was interpreted in terms of a dual singlet oxygen and radical mechanism, the radical contribution increasing with sensitiser in the order ER = MB < HP ≪ RF, but also showing some dependence on substrate unsaturation. Evidence is presented for singlet oxygen quenching by MB and RF ( k_O = 1.6 and 6.0 times 10⁷ dm³ mol^-1 s^-1) and for the accelerated photobleaching of the dye sensitisers in the presence of the unsaturated esters.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM

Abstract —Purple membrane suspensions change their color to blue and the absorption maximum shifts to 608 nm when the membrane is deionized on a cation exchange column or when it is washed first with < 2N NaCl followed by deionized water. The deionized chromophore is essentially identical with the chromophore produced by lowering the pH of the native membrane to < 4.0 (p K < 3.0). However, the deionized membrane does not aggregate and can be obtained in the pure state. The original purple color of the membrane is restored by addition of around 1 m M Na⁺, K⁺ or 10 μ M Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Pb²⁺ or La²⁺ when the protein concentration is 5μ M . The required salt concentrations decrease with decreasing pH. Direct measurement of bound Ca²⁺ by atomic absorption spectroscopy yields a ratio of Ca²⁺ to protein of <2 and a binding constant of 1.4 × 10⁶. Titration of the spectral change with salts at different pH values shows a linear relation between the pH and the logarithm of the salt concentration, with a 1:1 ratio for Na⁺ and 1:2 ratio for Ca²⁺. These relations are well predicted by Gouy-Chapman theory; however, the accompanying release of protons, changes of the CD spectrum, the complex kinetics of the spectral change during reconstitution with salt and preliminary X-ray diffraction results all suggest that conformational changes may be occurring in the protein.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE: A KINETIC STUDY OF ENDOGENOUS LIGHT-INDUCED FREE RADICALS IN REACTION CENTER PREPARATIONS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have detected, by means of a kinetic analysis, a rapidly decaying signal in reaction center preparations from the R26 blue-green mutant of Rhodopseudomonas spheroides. This signal, which we designate Signal B3, is essentially the same as that seen previously in iron-free preparations. Signal B3 decays at 20°C with a 1/ e time of ˜ 3 ms and exhibits an activation energy of 5 ± 1 kcal mol^-1 over the temperature range 0–30°C. Extraction with isooctane completely eliminates Signal B3, whereas readdition of exogenous ubiquinone-30 completely restores the signal. o -Phenanthroline has no effect on Signal B3. We discuss these results in terms of a model in which the primary acceptor is an iron-ubiquinone complex with excess ubiquinone serving as a secondary electron acceptor pool.