PHOTOINDUCED REDUCTION OF CYTOCHROME c IN THE PRESENCE OF HYDROPEROXIDE DERIVATIVE OF BIPHENYL

Abstract The photosensitive hydroperoxide derivative of biphenyl (BPP) was synthesized by the ozonolysis of phenanthrene in methanol. When cytochrome c (cyt c ) was illuminated by UVB light in the presence of BPP (BPPUV), it was reduced both under aerobic and anaerobic conditions. The action spectrum of the reduction was consistent with that of photolytic decomposition of BPP. Both gave maximum reactions at wavelengths around 300–310 nm. Electron spin resonance studies, using 5,5-dimethyl-1-pyrroline N -oxide as a spin-trapping reagent, revealed the generation of hydroxyl radicals in the BPPUV system. Product analysis of adamantane oxidation by BPPW also suggested the generation of hydroxyl radicals rather than singlet oxygen. However, the effects of scavengers were complicated. Singlet oxygen scavengers significantly inhibit the reaction while none of the hydroxyl radical scavengers tested was effective in inhibiting the BPPUV-mediated cyt c reduction. Deuterium oxide, which extends the lifetime of singlet oxygen, inhibited rather than enhanced the reaction. Reduction of cyt c was inhibited by salts, and their activities were correlated to the electron-donating nature of the anions. These results suggest that reduction of cyt c is mediated by electron transfer from a light-induced product of BPP, rather than by free hydroxyl radicals or singlet oxygen.     

REDOX PROTEINS AS ELECTRON ACCEPTORS FROM CHLOROPHYLL TRIPLET STATE IN LIPID BILAYER VESICLES: KINETICS OF REDUCTION OF MEMBRANE RECONSTITUTED CYTOCHROME c OXIDASE MEDIATED BY 2,5-DI-t-BUTYL BENZOQUINONE AND CYTOCHROME c

Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition.     

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVE BY TWO PHOTOREACTIONS. II. PHOTOCHEMICAL REGENERATION OF REDUCED CYTOCHROME c BY FLAVIN TRIPLET STATE AND ETHYLENEDIAMINETETRAACETIC ACID IN THE INNER COMPARTMENT AND REDUCTION OF FERREDOXIN BY CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

We have attempted to mimic natural photosynthesis with regard to the photogeneration of a powerful reductant, using a negatively charged lipid bilayer vesicle system incorporating two photoreactions sensitized by a flavin analog (flavin mononucleotide [FMN]) and chlorophyll (chl) in their respective triplet states. Ethylenediamine-tetraacetic acid (EDTA) in the inner aqueous compartment was used as a sacrificial electron donor to the FMN triplet, and ferredoxin in the outer aqueous compartment served as the final electron acceptor (mediated via triplet electron transfer chain in this multicomponent system to be elucidated. By itself, EDTA does not function as an effective donor to membran-bound oxidized chl (chl^+.), which is formed by electron transfer from triplet chl to the viologen follwed by transbilayer electron migration. This is a consequence of electrostatic repulsive interactions with the negatively charged membrane. This limitation is avoided when FMN is used as a photomediator between EDTA and chl^+.. The overall reaction is dramatically increased in rate by enclosing cytochrom c together with EDTA and FMN in the inner compartment. The rate constant of the key step in the reaction, i.e. elctron transfer from reduced cytochrome c, generated via photoreduction by the FMN/EDTA system, to chl^+. is increased 20-fold over that obtained with cytochrome c alone as the elctron donor. One of the important constraints that limited the net electron transfer across the bilayer to 50% of the added cytochrome, i.e. inhibition by oxidized cytochrome c formed in the inner compartment, is avoided by the inclusion of the second photoreaction in this system, thus allowing photoreduction of all of the added ferredoxin to be achieved. This system provides a model for a photochemical energy storage process that utilizes two photorections operating in series resulting in electron flow across a lipid bilayer membrane.     

THE REDUCTIVE PHOTOALKYLATION OF FLAVIN BY N-ALLYLTHIOUREA

Illumination of flavin in the presence of N-allythiourea (ATU) inhibits catalytic turnover of flavin between its reduced and oxidized redox states by adduct formation, the adduct being no longer reoxidisable by oxygen. The first step in the mechanism of adduct formation is an electron transfer from ATU to flavin in the photoexcited triplet state. In further steps, the ATU radical cation deprotonates, electronic rearrangement occurs and radical combination with the flavosemiquinone follows, yielding a cyclic product in which ATU is added in the 4a- and 5-position to the flavin chromophore.
Reaction rates and yields were determined by flash photolysis and continuous illumination. A photochemical study by variation of the molecular structure of ATU was undertaken to prove the proposed mechanism and to determine the structural requirements of flavin inactivators.     

KINETICS OF STACKING INTERACTIONS IN FLAVIN ADENINE DINUCLEOTIDE FROM TIME-RESOLVED FLAVIN FLUORESCENCE

The time dependence of the fluorescence of flavin adenine dinucleotide (FAD) was measured with a subnanosecond-resolving fluorometer. In contrast to the fluorescence decay of FMN, the decay of FAD was proved to be nonexponential. The time-dependent fluorescence of FAD can be interpreted by assuming an equilibrium between closed and open conformers in the ground state. The rate constant for folding in the excited state and the fluorescence lifetime of the intramolecular complex could be evaluated from analysis of the observed fluorescence decay. The results on FAD were compared to those on NADH obtained earlier.     

QUINONES AS MEDIATORS OF ELECTRON TRANSFER FROM MEMBRANE-BOUND CHLOROPHYLL TO CYTOCHROME c IN THE AQUEOUS PHASE IN LIPID BILAYER VESICLES: SYNERGISTIC ENHANCEMENT OF CYTOCHROME c REDUCTION BY LIPOPHILIC/ HYDROPHILIC QUINONE PAIRS

Laser flash photolysis has been used to determine the kinetics of cytochrome c reduction by chlorophyll triplet state in negatively-charged lipid bilayer vesicles, as mediated by quinones. Large synergistic enhancements in the yield of reduced cytochrome were obtained using a pair of quinones, one of which was lipophilic (e.g. benzoquinone, 2,6-di-f-butylbenzoquinone) and the other of which was hydrophilic (e.g. l,2-naphthoquinone-4-sulfonate). The mechanism was shown to involve initial quenching of the triplet by the membrane-associated quinone to form chlorophyll cation radical and quinone anion radical. An interquinone electron transfer process followed this reaction, which occurred at the membrane-water interface, and greatly facilitated electron transport from within the bilayer to the aqueous phase. This process formed the basis of the synergistic effect. Cytochrome c reduction occurred in the water phase by reaction with the anion radical of the hydrophilic quinone. Finally, the reduced cytochrome was reoxidized by a slow reaction with chlorophyll cation radical. Under the most favorable conditions, we estimate that the quantum yield of conversion of triplet quenching events to reduction of cytochrome approached unity. The lifetime of the reduced protein and oxidized chlorophyll could be as long as 140 ms, under the best conditions. This system has properties which are thus quite favorable for solar energy conversion in a biomimetic process.     

KINETICS and THERMODYNAMICS OF THE AUTOXIDATION OF REDUCED FLAVIN MONONUCLEOTIDE (FMN)

Abstract— Pulse radiolysis of flavin mononucleotide (FMN) solutions produce flavin semiquinone radicals. The equilibrium constant of radical formation was determined in the pH range2–7 as a function of flavin concentration. Several complex constants as well as the kinetics of equilibration were measured in this pH regime. The rate constant of the autoxidation of the free flavin mononucleotide semiquinone radical was determined to be (1 ± 0.5) × 10⁴ M ⁻¹ s⁻¹. It is shown that between pH 2 and 7 the direct reaction of the dihydroflavin with oxygen is negligibly slow compared to the rate of autocatalysis. The autoxidation of dihydroflavin is discussed in relation to electron transfer theory.     

钼铈氧化物体系的程序升温还原与还原动力学

运用X射线衍射、红外光谱、热重分析和比表面积测定等方法剖析了钼、铈氧化物的程序升温还原过程。结果表明，金属离子间的相互作用使得氧化物还原反应的活化能降低，有利于其催化性能的提高。     

TRANSIENT KINETICS OF FLAVOCYTOCHROME c PHOTOREDUCTION BY METHYL VIOLOGEN

Abstract— It has been demonstrated that photoreduced methyl viologen is stable during a flash photolysis experiment if a proper anaerobic environment is maintained. Therefore a new class of potential electron donors can be introduced in the study of transient phenomena associated with intermolecular electron transfer of redox proteins. The transient redox kinetic parameters of the flavocytochrome c from the photosynthetic bacterium Thiocapsa roseopersicina are reported for the first time and compared to similar data published for other fiavoproteins from diverse sources using flavin-type electron donors. The advantages of the photoreduced viologen based transient electron donor system are discussed.     

HDEHP萃取钙的动力学研究

近几年来萃取化学基础理论方面的研究不断深入,在广泛的热力学研究的基础上,萃取动力学的研究也日益增多。前文对HDEHP(二-(2·乙基已基)磷酸,用HA表示)萃钙的平衡与机理进行了研究,本文拟对Ca²⁺,NO₃⁼/HDEHP-萃体系萃取过程动力学加以探讨。     

PHOTOREDUCTION OF CYTOCHROME b551 OF PARTIALLY PURIFIED NEUROSPORA NITRATE REDUCTASE VIA ITS INTERNAL FLAVIN

Abstract— Nitrate reductases (NR) from NR-normal Neurospora crassa mutant albino band and from NR-defective mutantsnit–1 andnit–3 were isolated and partially purified in order to test the photo-reducibility of their cytochrome b ₅₅₇ via the NR-internal FAD. Photoreducibility with blue light of the isolated enzyme was observed as absorbance increase at 423, 524 and553–557 nm. It was independent of NADPH-nitrate reductase activity and could be induced if the dissociable FAD was not lost in the isolation procedure. The photoreduction of cytochrome b ₅₅₇ was readily reversible due the high autoxi-dation rate of this cytochrome. Therefore, anaerobic conditions are required for photoreduction with low light intensities. If aerobic conditions are applied, high intensities become necessary to overcome the simultaneous cytochrome h₅₅₇ oxidation.     

酸性条件下两种马来酸-烯烃交替共聚物诱导细胞色素c的结构转换

在pH=2·9时,细胞色素c保持类天然的结构;和马来酸-烯烃交替共聚物作用后,细胞色素c的α-螺旋结构基本保持不变,但是三级结构被破坏.另一方面,在pH=2·1时,细胞色素c去折叠形成伸展的无规卷曲构象;马来酸-烯烃交替共聚物可以诱导酸变性的细胞色素c从无规卷曲构象转变为α-螺旋结构.在酸性溶液中,由于马来酸-异丁烯交替共聚物和细胞色素c之间更强的相互作用,其对蛋白质结构的影响大于马来酸-1-十四烯交替共聚物.相对于小分子,聚合物可以在低浓度条件下提供有利于蛋白质结构转换的微环境.     

PHOTOREDUCTION OF HORSE HEART CYTOCHROME c

Abstract— During the course of studies on the mechanism of electron transport as catalyzed by cytochrome c , we have found that ferri-horse heart cytochrome c will undergo photoreduction. We have characterized the photoreduction of horse heart cytochrome c in regard to the photochemistry, the nature of the electron donor, and effect of solvent (pH, solvent perturbation, ionic strength). We conclude that ferri-horse heart cytochrome c undergoes photoreduction mediated by a light-induced heme excited state. Further, the protein moiety of cytochrome c serves in part to quench the formation of the excited state, and also to control the interaction of the electron donor and photo-excited cytochrome.     

SOME ONE ELECTRON REDUCTION PRODUCTS OF FLAVIN ANALOGS: CYANOISOALLOXAZINES AND DEAZAISOALLOXAZINES

Abstract— 8-Cyanoisoalloxazines have been previously shown to form highly stable radical species at basic pH. We have measured the electron spin resonance (EPR) spectra of the radical forms of 8-cyano-10-methyl-3-sulfopropylisoalloxazine (I) at both acidic and basic pH. In both cases. the EPR spectra are similar to those obtained from unsubstituted isoalloxazines. with no indication of hyperfine splitting due to the cyano nitrogen. Laser photolysis of I in the presence of EDTA at basic pH generates two radical species. One of these decays rapidly by a first-order process to produce thc stable radical. The rate of this decay depends upon the initial flavin concentration, thus suggesting a reaction of the radical with oxidized isoalloxazine. The rates of reaction of the radical species with added oxidants (O₂, ferricyanide), and the pH-dependence of stable radical formation, indicate that the rapidly-decaying species is the anion radical of I, and that the stable radical is formed by its reaction with oxidized flavin. Laser photolysis of I at acidic pH, as well as of 8-cyano-5-deaza-isoalloxazine at acidic or basic pH, does not generate stable radical species. I-Deazaisoalloxazines do not give radical transients at all upon laser photolysis.     

PHOTOSENSITIZED DEAMINATION OF SULFUR-AMINO ACIDS BY FLAVIN MONONUCLEOTIDE*

Abstract— S-Methylcysteine, methionine and cystine were found to be photochemically deaminated at a much higher rate than methionine sulfoxide, homomethionine and other non-sulfur amino acids in the presence of flavin mononucleotide. These data strongly support the view that the initial oxidation of those sulfur-amino acids by the photoactivated flavin is a one-electron abstraction from the sulfur atom followed by an intramolecular electron transfer from the carboxyl anion of the amino acid to the sulfinium radical, resulting in the decarboxylation-deamination.     

Pt/Al2O3蜂窝状催化剂上NO选择性催化还原反应动力学研究

采用多次涂层和浸渍法制备了蜂窝状Pt/Al2O3催化剂，并在高空速和大气体流量条件下对无梯度循环式反应器和积分反应器上催化剂的活性进行了比较。同时采用循环式反应器对动力学数据进行了测定。根据Langmuir-Hinshelwood模型和实验结果，推测了NO-C3H6-O2体系的SCR反应机理，并导出了NO和C3H6反应速率的数学表达式。据此所计算的理论模拟值能够与实验值很好地吻合。实验结果表明，氧气浓度对NO和C3H6的反应速率有明显的影响，二者均随着氧浓度c (O2)的增加达到峰值，再增加氧气浓度时，C3H6的反应速率r(C3H6)保持不变，而NO反应速率r (NO)却下降，而且下降的程度随着温度的升高而加剧。同时，随着氧气浓度增加，r (NO)达到最大值时的温度亦随之下降。     

RED LIGHT-INDUCED REDUCTION OF A PARTICLE-ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Abstract— In the presence of methylene blue, red light causes the reduction of a h-type cytochrome in particulate fractions from corn coleoptiles. Two types of difference spectra for the cytochromes in these fractions are presented: (a) red light-minus-dark in the presence of methylene blue, and (b) dithionite-reduced-minus-oxidized. Comparison of these spectra shows that photoexcited methylene blue selectively reduces a b-type cytochrome which constitutes at most only 30% of the total dithionite-reducible cytochrome present in the most active fractions. The photoreducible cytochrome has an alpha band at room temperature near 557 nm. Bleaching of methylene blue precedes cytochrome reduction under appropriate conditions, suggesting that the photoreduced dye is donating an electron to the cytochrome. This electron transfer does not involve a flavin, at least as judged by the absence of light-induced spectral changes attributable to flavins. Preliminary kinetic studies suggest that EDTA provides the pool of electrons for the reaction. The cytochrome cannot be assigned exclusively either to mitochondria or to endoplasmic reticulum, as judged by its sedimentation properties. These results and the current literature are discussed in the context of the hypothesis that this b-type cytochrome may be involved in the photoreception mechanism for blue and uv light in vivo.     

THE REDUCTION AND QUENCHING OF PHOTOEXCITED FLAVINS BY EDTA

Riboflavin was irradiated anaerobically in aqueous EDTA solutions over the pH range 2.5–10. In other dye systems (Bonneau and Pereyre, 1975), only the trivalent anion of EDTA was found to have significant reactivity for photoreduction. For riboflavin, the reactivity begins with monoanionic EDTA, and the reactivity is markedly increased as the charge increases. This suggests that the charge on the reductant is more important to the electron transfer process for riboflavin than the formation of a nonhydrogen bonded nitrogen site on EDTA. At high concentrations of EDTA in the pH range 4–8, quenching of the photoreduction occurs, which can be explained by an energy transfer between the excited singlet state of riboflavin and trianionic EDTA, possibly as an association complex. The rate constants for the photoreduction of riboflavin by the monovalent, divalent, and trivalent anions of EDTA are 1.0 times 10⁷M^-1 s^-l, 4.8 times 10′M^-1 s^-l, and 2.0 times 10⁸M^-1s^-1, respectively. The rate constant for the singlet state quenching by trianionic EDTA is 3 times 10⁹M^-l s^-1, and the limiting quantum yield for intersystem crossing for riboflavin in aqueous solution is 0.50 ± 0.05.     

二氧化硫脲还原沉析银的电势法研究

本文采用电势法研究二氧化硫脲的银氨溶液以沉析银的反应体系。获知体系温度、ｐＨ值、氨含量对反应速度和银沉析率均有较大的影响。在ＮＨ３和Ａｇ＾＋摩尔比为１８：１时，体系的最佳反应条件是：ｐＨ＝１２．００，温度为５０℃。在此条件下，将二氧化硫脲溶液不断地加入到［Ａｇ（ＮＨ３）２＾＋］溶液中，当反应体系的电势降到－７００ｍＶ（ｖｓ．ＳＣＥ）时，停止加入还原剂，并迅速中止反应，此时银的还原沉析率达９９．９％