A BLUE LIGHT-SENSITIVE CYTOCHROME-FLAVIN COMPLEX FROM CORN COLEOPTILES. FURTHER CHARACTERIZATION*

Abstract— The partially purified blue light-sensitive membrane-associated flavin-cytochrome complex from etiolated corn coleoptiles shows a unique sharp α-band at 555 nm in its light-minus-dark difference spectrum at liquid nitrogen temperature. This band is clearly distinguishable from the α-bands found in fractions enriched for mitochondria and endoplasmic reticulum respectively. The photoactive membrane fraction is shown to have ATPase activity that is not stimulated by K⁺ and that is not inhibited by oligomycin. Other than flavin fluorescence at 525 nm obtained upon excitation at 450 nm, there is a second fluorescent component with emission at 430 nm on excitation at 350 nm. The mid-point potential of the Triton X-100 solubilized b-cytochrome, measured by simultaneously monitoring the reduction of the pyocyanine 600-800 nm peak and the appearance of the 427 nm Soret peak of the b-cytochrome upon titration with dithionite in the presence of ferricyanide, is estimated to be ?65 mV. The kinetics of the blue light-induced reduction and dark rcoxidation of the 6-cytochrome suggest that the mid-point potential of the b-cytochrome is not affected by Triton X-100 solubilization.     

Intramolecular Electron Transfer in CO-Bound Mixed-Valence Cytochrome c Oxidase Following CO Photolysis

Internal electron transfer in bovine cytochrome c oxidase was initiated by CO photolysis of the CO-bound mixed-valence form of the enzyme. Transient absorption spectroscopy was used to monitor changes in the redox states of the metal centers in the enzyme brought about by electron re-equilibration. Upon CO photodissociation, reduced high spin cytochrome a₃ was generated in less than 0.1 μsec, and a portion of the reduced cytochrome a₃ was reoxidized with biphasic rate constants of k₁ = 1.0 × 10⁶ s^?1 and k₂ = 7.8 × 10⁴ s^?1. Concomitant reduction of cytochrome a was also observed with biphasic rate constants of k₁ = 1.6 × 10⁶ s^?1 and k₂ = 9 × 10⁴ s^?1. The stoichiometry of cytochrome a₃ oxidized to cytochrome a reduced was found to be close to 1:1. Contrary to similar studies in the literature, no reduction of Cu_A was observed. As a control, no transient absorption changes corresponding to electron transfer was observed in the CO-inhibited fully reduced form of the enzyme. These results indicate that there is significant electron reequilibration only between cytochrome a₃ and cytochrome a upon photolysis of the CO-bound mixed-valence enzyme.     

PHOTOCHEMISTRY OF THE PHOTOTOXIC POLYACETYLENE PHENYLHEPTATRIYNE*

Abstract— Investigation of the photochemistry of the phototoxic polyacetylene phenylheptatriyne, PHT, was undertaken to obtain further information on competing photo-oxidative type II and non-oxidative processes observed in vivo. Laser excitation (308 or 337 nm) led to the formation of a strong triplet signal with a lifetime of 28 μs in MeOH. The triplet was efficiently quenched by the triplet quencher 1,3-octadiene (k_q - 1.6 × 10⁹M^-1s^-1). Quenching by O₂ occurred with a rate constant (1.7 × 10⁹M^-1s^-1) comparable to the rate of electron transfer to methylviologen (1.4 × 10⁹M^-1 s^-1). The formation of singlet oxygen established earlier (type II reaction) in the former case and the semioxidized PHT radical in the latter case are consistent with the competing phototoxic processes observed in vivo.     

ELECTRON TRANSFER REACTIONS OCCURRING UPON LASER FLASH PHOTOLYSIS OF PHOSPHOLIPID BILAYER SYSTEMS CONTAINING CHLOROPHYLL,BENZOQUINONE AND CYTOCHROME c-I. NEGATIVELY-CHARGED VESICLES*

Abstract— In negatively-charged lipid bilayer vesicles prepared in deionized water from egg phosphatidylcholine and 25 mol % of α-eleostearic acid, and containing chlorophyll a, benzoquinone, and cytochrome c, primary electron transfer after a laser flash occurred principally from chlorophyll triplet to benzoquinone, and to a smaller extent from chlorophyll triplet to oxidized cytochrome c. Several secondary electron transfer reactions occurred subsequent to this. The most rapid of these was electron transfer from reduced cytochrome c, which was bound to the outer surface of the negatively-charged vesicle, to chlorophyll cation radical (k= 3.9 times 10³ s^-1). Subsequent to this, the cation radical was reduced by benzoquinone anion radical (k= 1.6 times 10² s^-1>) and bound oxidized cytochrome c was reduced by the remaining anion radical which was expelled into the aqueous phase by the negative charge on the vesicle surface. This latter reaction occurred at the membrane-solution interface with an observed rate constant (k= 60 s^-1) two orders of magnitude smaller than cytochrome oxidation. Net reduced cytochrome c was produced in this process. The reduced cytochrome c was slowly reoxidized by benzoquinone (k= 17 s^-1) and the system was returned to its original state. When the vesicle system was made slightly basic by adding tris(hydroxymethyl)aminomethane, the rates of both the reverse electron transfer between chlorophyll cation radical and benzoquinone anion radical (k= 5 times 10² s^-1) and the oxidation of reduced cytochrome c by chlorophyll cation radical (k= 9.4 times 10³ s^-1) were accelerated. The rate of reduction of oxidized cytochrome c by benzoquinone anion radical remained approximately the same.     

Electron transfer at tetrahedral cobalt(II), part IV: kinetics of silver(I) catalysed chlorate reduction

Summary The very slow reduction of ClO _inf3 ^su– by [CoW₁₂O₄₀]^6- is markedly catalysed by Ag¹ in aqueous HClO₄ solution at I = 1.0 M (NaClO₄). The reaction obeys the rate law: -d[reductant]/dt where a = (1.28 ± 0.10) × 10^-4M^-1s^-1 and b = (2.22 ± 0.20) × 10^-4M^-3s^-1, at T = 60.1 ± 0.1°C and [H⁺] = 0.10–1.50 M. Activation parameters have been determined for each pathway and the reaction is discussed in terms of the outer-sphere mechanism.Part III: ref. 1.     

Reactive oxygen and nitrogen species (RONS) scavenging reactions of o-vanillin: Pulse radiolysis and stopped flow studies

Reactions of peroxyl radicals and peroxynitrite with o-vanillin (2-hydroxy 3-methoxy benzaldehyde), a positional isomer of the well-known dietary compound vanillin, were studied to understand the mechanisms of its free radical scavenging action. Trichloromethylperoxyl radicals (CCl₃O ₂ ^· ) were used as model peroxyl radicals and their reactions with o-vanillin were studied using nanosecond pulse radiolysis technique with absorption detection. The reaction produced a transient with a bimolecular rate constant of approx. 10⁵ M⁻¹s⁻¹, having absorption in the 400–500 nm region with a maximum at 450 nm. This spectrum looked significantly different from that of phenoxyl radicals of o-vanillin produced by the one-electron oxidation by azide radicals. The spectra and decay kinetics suggest that peroxyl radical reacts with o-vanillin mainly by forming a radical adduct. Peroxynitrite reactions with o-vanillin at pH 6.8 were studied using a stopped-flow spectrophotometer. o-Vanillin reacts with peroxynitrite with a bimolecular rate constant of 3 × 10³ M⁻¹s⁻¹. The reaction produced an intermediate having absorption in the wavelength region of 300–500 nm with a absorption maximum at 420 nm, that subsequently decayed in 20 s with a first-order decay constant of 0.09 s⁻¹. The studies indicate that o-vanillin is a very efficient scavenger of peroxynitrite, but not a very good scavenger of peroxyl radical. The reactions take place through the aldehyde and the phenolic OH group and are significantly different from other phenolic compounds.     

Electron transfer,equilibrium, and protonation in the system of cis- and trans-stilbene in 2-propanol

Spectrophotometric pulse radiolysis experiments with cis- and trans-stilbene (S_c and S_t) in 2-propanol show that both isomers react with the solvated electron with a rate constant of 4.5 × 10⁹ M^?1 s^?1. The absorption spectra of the two anion radicals have maxima at 496 and 486 nm, respectively. The absorbances at 400–550 nm disappear exponentially corresponding to a pseudo first order protonation of the anion radicals. The rate constants for the protonation of the cis isomer is 6.4 × 10⁵ and of the trans isomer 0.7 × 10⁵ s^?1. In mixtures of cis- and trans-stilbene the electron transfer

has a forward rate constant of 9 × 10⁷ M^?1 s^?1 while the back reaction has a rate constant of 2.15 × 10⁷ M^?1 s^?1. An equilibrium constant K = 4.2 is calculated.     

LIGHT-INDUCED ABSORBANCE CHANGES IN CHLOROPLASTS AT -196°C*

Abstract— Absorbance changes induced by irradiating chloroplasts at — 196°C were measured in the region of 525–575 nm with a single-beam spectrophotometer. Irradiation at low temperature caused a bleaching at 556 nm due to oxidation of cytochrome b₅₅₉ but little or no change of cytochrome f. There occurred in addition a loss of absorbance at 547 nm and an increase at 543 nm. The bleaching at 547 nrn (and possibly the increase at 543 nm) could be induced chemically with dithionite or borohydride but not ascorbate. Subchloroplast particles with only Photosystem I activity showed no light-induced absorbance changes, while particles containing combined Photosystem I and Photosystem II activities showed the same changes as whole chloroplasts. Scenedesmus mutant No. 11 cells showed no absorbance changes while mutant No. 8 and wild-type cells showed the normal changes. It is concluded that the photooxidation of cytochrome b₅₅₉ and the photoreduction causing the bleaching at 547 nm are both mediated by Photosystem II.     

A PREVIOUSLY UNREPORTED INTENSE ABSORPTION BAND AND THE pK, OF PROTONATED TRIPLET METHYLENE BLUE

Abstract— Excitation by a Q-switched giant ruby laser (1.2 J output at 694 nm ?50 ns flash) of 2–10 µM solutions of methylene blue in water, 30% ethanol in water or 50 v/v% water-CH₃CN at pH values in the range 2.0–9.3 converted the dye essentially completely to its T₁ state. The absorption spectrum of T₁ dye was measured in different media at pH 2.0 and 8.2 by kinetic spectrophotometry. Previously reported T-T absorption in the violet in acidic and alkaline solutions and in the near infrared in alkaline solution was confirmed. Values found for these absorptions in the present work with 30% ethanol in water as solvent are λ_max - 370nm, ε_max, - 13,200 M^-1 cm^-1 at pH 2 and λ_max,?420nm, ε_max 9000 M^-1 cm^-1, λ_max, - 840 nm, ε_max - 20,000 m ^-1 cm^-1 at pH 8.2. Long-wavelength T-T absorption in acidic solution is reported here for the first time: λ_max, ? 680 nm, e_max? 19,000 M cm^-1 in 30% ethanol in water at pH 2. Observation of a pH-independent isobestic point ? 720 nm confirms that the long-wavelength absorptions are due to different protonated states of the same species, MB⁺(T₁) and MBH²⁺(T₁). The pK_a of MBH²⁺(T₁) in water was determined from the dependence on pH of absorption at 700 and 825 nm to be 7.1₄± 0.1 and from the kinetics of decay of triplet absorption to be 7.2. The specific rate of protonation of MB⁺(T₁) by H₂PO₄ in water at pH 4.4 was found to be 4.5 ± 0.4 times 10⁸M^-1s^-1.     

Complex dynamical behavior in the oxidation of hydroxylamine by bromate

Complex dynamical behavior has been observed in the oxidation of hydroxylamine by bromate in acidic sulfate medium. The reaction shows clock type kinetics in closed conditions and an aperiodic oscillations if gaseous products are removed from the system with a constant flow-rate. The reduction kinetics of bromate ions with excess hydroxylamine has been studied in the presence of allyl alcohol. The observed pseudo-first-order rate constant k_obs has been found to follow the expression where [hydroxylamine] is total initial hydroxylamine concentration, K₁ = 0.5 M^?1, K₂ = 10⁶ M^?1, and k = 2.57 × 10³ M^?1 s^?1 at 298.15 K and I = 2.0 M. The rate constant for the bromine oxidation of hydroxylamine in sulfuric aqueous solution has been determined. © 1994 John Wiley & Sons, Inc.     

Influence of cyanide and sulphide ions on the reduction and reoxidation of cobalt(II) in thiocyanate solution at mercury electrodes

The process of reduction and reoxidation of cobalt(II) in thiocyanate solution at hanging mercury drop electrode has been investigated by cyclic voltammetric, chronoamperometric and anodic stripping methods. In 0.1 M NaSCN and 0.4 M NaClO₄ solution containing 1×10^?3M cobalt(II), the voltammogram on the first cycle at 0.05 V s^?1 gives a cathodic peak at ?1.06 V with hysteresis on reversal, and an anodic wave with a peak potential of ?0.28 V and with two shoulders near ?0.38 and ?0.45 V, respectively. Multicyclic voltammograms under the same conditions give a cathodic peak at ?0.90 V and an anodic peak at ?0.45 V. The reduction and reoxidation of cobalt(II) in thiocyanate solution is accelerated by the reduction products of thiocyanate ion, cyanide and sulphide ions, which are produced during the electroreduction of cobalt(II).A mechanism of reduction and reoxidation of cobalt(II) which involves a chemical reduction of thiocyanate ion by electroreduced metallic cobalt and takes into account cyanide and sulphide ions is proposed. The hysteresis on the cathodic wave is caused by the difference in reduction potentials of cobalt(II)-thiocyanate and-cyanide complexes. Cyclic voltammetric study of cobalt(II) in perchlorate solution containing trace amounts of cyanide and sulphide ions supports these conclusions.     

Couloamperometry. A fast kinetic technique for halogenations. I. Bromination rate constants of highly reactive enol ethers

A new couloamperometric apparatus has been designed to extend the range of this kinetic technique to the measurement of very high rate constants, 10⁸M^?1s^?1, by using TFCR-EXSEL conditions (TFCR—very low reactant concentration; EXSEL—salt excess), which give half-lives of a few seconds for very fast second-order reactions. Very low faradaic currents, in the nanoampere range for halogens, corresponding to very low reactant concentrations of 10^?8–10^?9M, are measured selectively by compensating the eddy currents, principally the residual and the induced currents. When the electroactive species is bromine, the concentration is demonstrated to be linearly related to the limiting reduction current in the very low concentration range. The upper limit of this technique for bromination is at present 3 × 10⁸M^?1s^?1. The method is applied to the kinetic study of highly reactive enol ethers EtO-C(R) = CH-R′, where R and R′ are H or Me. A value of 2.2 × 10⁸M^?1s^?1 is obtained for k, the rate constant for free bromine addition to EtO-CH = CH₂, by extrapolating the kinetic bromide ion effects to [Br^?] = 0. An α-methyl effect (k_α-Me/k_H)_EtO of 15 is found; this is a small decrease in the methyl effect compared to the marked increase in the double bond reactivity. For the enol acetate MeCOO-CH = CH₂, whose rate constant is 6 × 10²M^?1s^?1, (k_α-Me/k_H)_OCOMe is 21. The dependence of substituent effects on reactivity is discussed in terms of the Hammond effect on the transition state position and of charge delocalization by group G of olefins G-CH = CH₂.     

Redox chemistry of o- and m -hydroxycinnamic acids: A pulse radiolysis study

Radiation chemical reactions of^•OH, O^•−, N₃ ^•and e _aq ^t- witho- and m-hydroxycinnamic acids were studied. The second-orderrateconstantsforthereaction of^•OH with ortho and meta isomers in buffer solution at pH7 are 3.9±0.2 × 10⁹ and 4.4 ± 0.3 × 10⁹ dm³ mol^-1 s^-1 respectively. At pH 3 the rate with the ortho isomer was halved (1.6 ± 0.4 × 10⁹ dm³ mol^-1 s^-1) but it was unaffected in the case of meta isomer (k = 4.2±0.6 × 10⁹dm³mol^-1 s^-1). The rate constant in the reaction of N₃ ^• with the ortho isomer is lower by an order of magnitude (k = 4.9 ± 0.4 × 10⁸ dm³ mol^-1s^-1). The rates of the reaction of e _aq ^t- with ortho and meta isomers were found to be diffusion controlled. The transient absorption spectrum measured in the^•OH witho-hydroxycinnamic acid exhibited an absorption maximum at 360 nm and in meta isomer the spectrum was blue-shifted (330 nm) with a shoulder at 390 nm. A peak at 420 nm was observed in the reaction of O^bb−with theo-isomer whereas the meta isomer has a maximum at 390 and a broad shoulder at 450 nm. In the reaction of the absorption peaks were centred at 370–380 nm in both the isomers. The underlying reaction mechanism is discussed.     

THE PHOTOEXCITED TRIPLET STATE OF TETRAPHENYL CHLORIN, MAGNESIUM TETRAPHENYL PORPHYRIN AND WHOLE CELLS OF CHLAMYDOMONAS REINHARDI. A LIGHT MODULATION-EPR STUDY

Abstract— The photoexcited triplet states of frozen solutions of tetraphenyl chlorin (TPC), magnesium tetraphenyl porphyrin (MgTPP) and whole cells of Chlamydomonas reinhardi have been studied by light modulation-EPR spectroscopy. The porphyrins were chosen to be studied as model compounds for chlorophyll molecules, From EPR spectra the zero field splitting parameters (ZFS) were calculated. For TPC, |D| = 0.0364 ± 0.0002 cm^-1, |E| = 0.0063 ± 0.0002 cm^-1. For MgTPP, |D| = 0.0310 ± 0.0002 cm^-1. For chloroplasts, |D| = 0.0280 ± 0.0004 cm^-1, |E| = 0.0032 ± 0.0004 cm^-1. In all compounds studied, except MgTPP, electron spin polarization (ESP) was observed. From the analysis of the kinetic curves at each canonical orientation we evaluated the spin lattice relaxation rate W, the depopulation rate constants k_p, and the ratio between the population rate constants, A_p, at zero magnetic field. For TPC in ethanol-toluene (5:1) k_x= (0.70 ± 0.10) × 10³ s^-1, k_y= (0.40 ± 0.07) × 10³ s^-1, k_x= (0.24 ± 0.05) × 10³ s^-1; A_x:A_y:A_z? 1.0:0.6:0.4; W= (2.60 ± 0.40) × 10³ s^-1. For MgTPP, only the total decay rate constant, k_T, was calculated: (1.5 ± 0.2) × 10 s^-1 in n-octane and (4.8 ± 0.8) × 10 s^-1 in ethanol. The results for TPC and MgTPP are compared to those reported previously for chlorophyll. It is concluded that the dynamics of the photoexcited triplet state in chlorophylls are mainly governed by the chlorin macrocycle. From the EPR spectrum and ZFS parameters of chloroplasts, we propose that both chlorophyll a and chlorophyll b are the main constituents of the EPR spectrum. From the analysis of the kinetic curves we obtain separately the kinetic parameters for chlorophylls a and b, k^a_x= (1.30 ± 0.20) × 10³ s^-1, k^a_y;= (0.85 ± 0.15) × 10³ s^-1k^a_x= (0.32 ± 0.05) × 10³ s^-1; A^a_x:A^a_y:A^a_z? 1.0:0.7:0.2; W^a= (1.20 ± 0.20) × 10³ s^-1; k^b_x= (0.56 ± 0.09) × 10³ s^-1, k^b_y= (0.30 ± 0.04) × 10³ s^-1, k^b_z= (0.06 ± 0.01) × 10³ s^-1; A^b_x:A^b_y:A^b_x? 1.0:0.6:0.1; W^b= (5.00 ± 0.80) × 10³ s^-1. These results are very close to those found separately for chlorophyll a and chlorophyll b oligomers in vitro.     

Der Einfluß großer Salzkonzentrationen auf die Oxydationsreaktion von Fe(phen) 3 2+ mit Ce(IV) in Schwefelsäure

The influence of NaClO₄, NaCl and Na₂SO₄ on the oxidation of Fe(phen) ₃ ²⁺ by Ce(IV) was investigated by means of the stopped-flow method. At the concentrations range of NaClO₄ and NaCl 0.1–1.0M the rate constant values decrease from 1.03·10⁵ to 0.56·10⁵M^–1s^–1 and from 1.08·10⁵ to 0.81·10⁵M^–1s^–1 respectively.In varying concentrations of Na₂SO₄ solutions (0.05–0.35M) the rate constant values decrease from 1.05·10⁵M^–1s^–1 to 0.45·10⁵M^–1s^–1.Taking into account the negative salt effect the mechanism of the reaction progress is proposed.

     

Thiocyanate-assisted demetallation of α,β,γ,δ-tetra(p-sulfophenyl)porphinatoindium(III) in aqueous media

The rate law for the demetallation of the title indium(III)-porphin complex in aqueous acidic thiocyanate media at 3.00M ionic strength was found to be of the form where [H₄P^2?] is the concentration of the diacid product formed, [InP]_t is the total concentration of all forms of indium(III)-porphin complex present, and a and b are constants. The constant a is a pseudo-third-order rate constant with the value (0.057 ± 0.005)M^?2 s^?1 and b has the value 0.704M^?2 at 50.5°C. If the mechanism for demetallation involves ringpuckering with the attachment of two H⁺ ions, then 1/b can be identified with the product K₁K₂ for the stepwise dissociation of two protons from two ring pyrrolic nitrogen atoms of H₂InP^?. In the sulfonated tetraphenylporphin used for these studies the ring pyrrolic nitrogen atoms seem to be the most probable sites for protonation. If this identification is correct, the value of 1.42 ± 0.13 found for the product K₁K₂ shows the enormous effect that the presence of the In³⁺ center has on the ionization constants of these two protons. That the kinetic studies show saturation effects with respect to proton addition to InP^3? may result from the fact that In³⁺ sits about 0.6 Å above the porphin ring.     

The mechanism of the electrochemical reduction of 5-nitrouracil

The electrochemical reduction of 5-nitrouracil (5NU) has been investigated on Ag-Hg cathode in DMSO by cyclic voltammetry and controlled potential electrolysis, in combination with UV and ESR spectroscopies. The results showed that 5NU proceeds in a four-electron reduction and simultaneously participates in the reaction as a proton donor. At a more negative potential, the 5NU anion (5NU^?) thus formed is reduced further through a one-electron process to a dianion whose ESR parameters have been determined as a_N = 14.6 G, a_H = 5.2 G and g = 2.005. The radical decay process might be conjectured through protonization by accepting protons from 5NU, of which the rate constant k₁ = 52 M^?1·s^?1.     

COMPLEX FORMATION BETWEEN PYRENE AND THE NUCLEOTIDES GMP, CMP, TMP AND AMP

Pyrene has been found to form ground and excited electronic state complexes of 1:1 stoichi-ometry with GMP, CMP, TMP and AMP. The values of their ground state association constants are 45 M^-1, 13M^-1, 14 M^-1, and 52 M^-1 respectively. The fluorescence of pyrene is strongly quenched by GMP, CMP, and TMP but only slightly by AMP. Fluorescence quenching analysis has yielded the values 87M^-1, 73 M^-1, and 154 M^-1 for the excited state association constants with GMP, CMP, and TMP, respectively. The corresponding values for the excited state second-order rate constant for complex formation are: 3.3 times 10⁹M^-1 s^-1 4.1 times 10⁹M^-1 s^-1, and 4.0 times 10⁹M^-1 s^-1. The probabilities of complex formation per collision between an excited pyrene molecule and a nucleotide are: 0.52, 0.64, and 0.63. The values for the excited state rate constant for dissociation of the complex are: 3.8 times 10⁷s^-1 5.6 times 10⁷s^-1, and 2.6 times 10⁷s^-1. The possibility is discussed that partial transfer of charge from pyrene to nucleotide may be playing a role in the complex formation process.     

Rates of addition of tert-butyl and pivaloyl radicals to acrylonitrile measured by modulated ESR and μSR spectroscopy

For the rate constant of addition of tert-butyl radicals to acrylonitrile at T = 300 K in solution modulated ESR spectroscopy and muon spin rotation yield 10⁶ M^?1 s^?1 and 2.4 × 10⁶ M^?1 s^?1. The addition of pivaloyl radical to acrylonitrile proceeds with Arrhenius parameters log A/M^?1 s^?1 = 7.7 and E_a = 11.5 kJ/ mol. The results are discussed in terms of polar effects in radical addition reactions.