Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

Kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II

The kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II has been studied over the pH range 5.2-9.9 at 25 degrees C and an ionic strength of 0.11 M. For all pH values, exponential decay curves are obtained for the reaction of compound II in the presence of ferrocyanide which yielded pseudo-first-order rate constants kobs. The spontaneous decay of compound II in the absence of ferrocyanide occurs at an appreciable rate which was measured independently and used in the data analysis. At all pH values two striking effects were observed when the rate of the decay reaction in the presence of ferrocyanide, kobs, was plotted against ferrocyanide concentration: a saturation effect and positive intercepts which are attributable to the spontaneous decay. The plots of kobs versus ferrocyanide concentration were analyzed in terms of the following parameters: a first-order rate constant k3,obs, a Michaelis constant Km,obs and a spontaneous-decay rate constant k4. The parameters k3,obs and Km,obs describe the reaction of compound II with ferrocyanide, independently of the spontaneous decay. The parameter k4 has only a small pH dependence, whereas plots of the logs of k3,obs and Km,obs versus pH have slopes of -1 at high pH. The major part of the pH dependence can be explained by the influence of a single heme-linked acid group in the LPO-compound-II-ferrocyanide complex.     

Photoinitiated ion movements in bilayer membranes containing magnesium octaethylporphyrin.

A photocurrent produced by planar lipid bilayers containing Mg-octaethylporphyrin in the presence of oxygen has been investigated to determine if the current is due to movement of the MgOEP+ ion in the bilayer. Photoexcitation of the MgOEP is known to produce MgOEP+ in the bilayer when an electron acceptor is present. However, the aqueous electron acceptors ferricyanide and methyl viologen (MV+2) have opposite effects on the photocurrent. Ferricyanide decreases the photo current, even in the presence of oxygen, whereas methyl viologen increases the photocurrent, but only when oxygen is present. We attribute most of the photocurrent to the movement of superoxide anion. The difference in effect between ferricyanide and methyl viologen is attributed to the different rates of reduction of O2 by reduced MV+ (fast) vs. ferrocyanide (slow) and the known competition between ferricyanide and oxygen as the acceptor for the photoexcited porphyrin. It is inferred that most of the MgOEP is localized in the polar region of the lipid bilayer. Addition of ferrocyanide to the aqueous phase on one side of the bilayer, to trap MgOEP+ produced on the other side by MV+2, fails to increase the lifetime of the photovoltage. With a pH gradient across the bilayer, we observed only 5% of the photovoltage expected for the selective transport of H+ or OH- by MgOEP+. Thus, these measurements set the lower limit for the cross bilayer transit time of MgOEP+ or its charge in the range of 0.1-0.5 s.     

Studies on the Hill reaction of membrane fragments of blue-green algae IV. Garotenoid photobleaching induced by photosystem II action

Carotenoid photobleaching induced by the action of photosystemII was investigated with membrane fragments of Anabaena cylindricaand A. variabilis. Carotenoid photobleaching occurred only when ferricyanide and/orCCCP was present in the reaction mixture. Maximum velocity ofthe reaction was obtained in the presence of ferricyanide andCCCP. Difference spectra (light minus dark) indicated that afast carotenoid photobleaching was accompanied by a slow chlorophyllbleaching. The pattern of the difference spectra was identicalto that reported by Yamashita et al. (1) with spinach chloroplasts.The reaction was DCMU-sensitive, though a portion of the activitywas insensitive to DCMU when ferricyanide was present in thereaction mixture. The effect of CCCP on stimulation of carotenoidphotobleaching showed the same function against CCCP concentrationas that on inhibition of DPIP-photoreduction with DPC. Carotenoidphotobleaching was stimulated by ferricyanide and suppressedby ferrocyanide; thereaction rate was reduced fifty percentwith a ferri- and ferrocyanide mixture giving 510 mV redox potential(pH 7.5). Benzoquinone was stimulatory, but DPIP had no effect. Incubation of membrane fragments in a dilute buffer inactivatedthe Hill reaction but neither the DPC-supported DPIP-photoreductionnor carotenoid photobleaching in A. variabilis. In A. cylindrica,incubation inactivated all three reactions. Inactivation ofthe latter two reactions followed the same kinetics. (Received August 31, 1972; )     

THE REACTIONS OF DENATURED EGG ALBUMIN WITH FERRICYANIDE

The following facts have been established experimentally. 1. In the presence of the synthetic detergent, Duponol PC, there is a definite reaction between dilute ferricyanide and denatured egg albumin. 0.001 mM of ferrocyanide is formed by the oxidation of 10 mg. of denatured egg albumin despite considerable variation in the time, temperature, and pH of the reaction and in the concentration of ferricyanide. 2. If the concentration of ferricyanide is sufficiently high, then the reaction between ferricyanide and denatured egg albumin in Duponol solution is indefinite. More ferrocyanide is formed the longer the time of reaction, the higher the temperature, the more alkaline the solution, and the higher the concentration of ferricyanide. 3. Denatured egg albumin which has been treated with formaldehyde or iodoacetamide, both of which abolish the SH groups of cysteine, does not reduce dilute ferricyanide in Duponol PC solution. 4. Cysteine is the only amino acid which is known to have a definite reaction with ferricyanide or which is known to react with dilute ferricyanide at all. The cysteine-free proteins which have been tried do not reduce dilute ferricyanide in Duponol PC solution. 5. Concentrated ferricyanide oxidizes cystine, tyrosine, and tryptophane and proteins which contain these amino acids but not cysteine. The reactions are indefinite, more ferrocyanide being formed, the higher the temperature and the concentration of ferricyanide. 6. The amount of ferrocyanide formed from denatured egg albumin and a given amount of ferricyanide is less in the absence than in the presence of Duponol PC. 7. The amount of ferrocyanide formed when denatured egg albumin reacts with ferricyanide in the absence of Duponol PC depends on the temperature and ferricyanide concentration throughout the whole range of ferricyanide concentrations, even in the low range of ferricyanide concentrations in which ferricyanide does not react with amino adds other than cysteine. The foregoing results have led to the following conclusions which, however, have not been definitely proven. 1. The definite reaction between denatured egg albumin in Duponol PC solution and dilute ferricyanide is a reaction with SH groups whereas the indefinite reactions with concentrated ferricyanide involve other groups. 2. The SH groups of denatured egg albumin in the absence of Duponol PC react with iodoacetamide and concentrated ferricyanide but they do not all react rapidly with dilute ferricyanide. 3. Duponol PC lowers the ferricyanide concentration at which the SH groups of denatured egg albumin react with ferricyanide. The SH groups of denatured egg albumin, however, are free and accessible even in the absence of Duponol PC.     

Relationship between Electron Transport across the Plasmalemma and a pH Decrease in the Bulk Medium

The hypothesis is tested that acidification of the bulk medium during transplasmalemma electron transport to ferricyanide is due solely to a requirement for charge balance. According to this hypothesis, reduction of the trivalent anion, ferricyanide, to the tetravalent anion, ferrocyanide, results in a charge difference that is balanced by protons. A coulometric device is used that rapidly and efficiently reoxidizes ferrocyanide to ferricyanide, thus maintaining a constant charge in the bulk medium. Oat (Avena sativa L. cv Garry) mesophyll protoplasts are chosen as experimental material to facilitate ferricyanide reduction and the concomitant ferrocyanide reoxidation by the coulometric device. The kinetics of ferricyanide reduction and proton excretion by protoplasts are similar to those of other cell types and tissues. Rates of net proton excretion are identical regardless of whether the ferrocyanide is simultaneously reoxidized. We conclude that acidification may occur during transplasmalemma electron transport when there is no change in negative charge of the bulk medium.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

Diffusion- and reaction rate-limited redox processes mediated by quinones through bilayer lipid membranes.

The mediation of redox reactions through bilayer lipid membranes was studied. With an appropriate choice of electron acceptors the redox process can be limited either by the chemical reaction rate between the mediator and the reactants or by the shuttle frequency of the mediator through the membrane. Both modes were demonstrated for redox reactions mediated by 2,6 dichlorobenzoquinone (DCBQ) and by alpha-tocopherol with ascorbate entrapped inside vesicles using ferricyanide (a mild oxidant) or hexachloroiridate (a strong oxidant) in the external solution. The redox processes were reaction rate-limited and diffusion-limited for ferricyanide and hexachloroiridate, respectively. The kinetics of the redox processes in the diffusion- and the reaction rate-limited modes allows the determination of the shuttle frequencies and of the interfacial reaction rates of the mediators, respectively. The shuttle frequencies of DCBQ and alpha-tocopherol were approximately 8 and 0.08 s-1, respectively, in L-alpha-dipalmitoyl phosphatidylcholine (DPPC) cholesterol vesicles at 25 degrees C. Interfacial reaction rates between the mediators and ferricyanide were about two- and tenfold lower compared with bulk reaction rates for DCBQ (water) and tocopherol (50% ethanol solution), respectively, i.e., tocopherol is relatively less accessible to aqueous oxidants at the membrane interface. Tocopherol and oxidized tocopherol are reversible hydrophobic redox couples that interact very rapidly with strong oxidants. In both modes of mediation DCBQ was more effective than alpha-tocopherol.     

Evidence for the extracellular reduction of ferricyanide by rat liver. A trans-plasma membrane redox system

1. Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. 2. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22mumol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide+ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. 3. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3mumol/min per g of cells proceeded for approx. 3min and derived from low-affinity sites (apparent K(m)>1.3mm). The secondary rate of 0.29mumol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent K(m)0.13mm). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. 4. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent K(m) for ferrocyanide oxidation by hepatocytes was 1.3mm. 5. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1mm-KCN but at a rate less than that of the reduction of ferricyanide. 6. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1mum-rotenone and 0.5mm-ferrocyanide, and stimulated by 0.1mm-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN(3), or p-hydroxymercuribenzoate but was inhibited by 2.6mm-CaCl(2), 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. Overall, these results are consistent with the occurrence of a trans-plasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Thiosemicarbazide-ferricyanide reduction for the histochemical demonstration of aldehydes in tissue sections

Aldehydes produced from carbohydrates by oxidation or acid hydrolysis may be visualized by application of aqueous thiosemicarbazide followed by Schmorl's ferricyanide reduction. The thiosemicarbazide reacts with the aldehydes by its hydrazine group, while its thiocarbamyl group remains active. The thiocarbamyl moiety is a strong reducing group that converts ferricyanide to ferrocyanide in Schmorl's reaction. The ferrocyanide is trapped immediately by the ferric salt, which deposits Prussian blue at the site of the aldehydes thereby demonstrating the location of the original substance.     

The mechanism of the ferric ferricyanide reduction reaction

Synopsis The ferric ferricyanide reaction has been generally considered to depend on the reduction of ferricyanide to ferrocyanide and, in the presence of the ferric ion, the consequent production of Prussian Blue. In testing the ability of ferricyanide to prevent the azo-coupling reactions of enterochromaffin cells and of the noradrenaline islets of the adrenal medulla, ferricyanide proved to be an effective oxidant in alkaline solution, but failed to quinonize the two catechols below pH 6. Similar results are obtained in oxidative blocking of cutaneous sulphydryl sites against the ferric ferricyanide reaction and against staining by the mercaptide acid azo-coupling reaction of Lillie & Glenner (1965).Ferric ferricyanide solutions are used at pH 2–2.5 and the ferric ion is an effective quinonizing and thiol-oxidizing agent at this pH level; the ferricyanide ion is not. The ferric ferricyanide reaction depends on the reduction of ferric to ferrous ions in the presence of ferricyanide, with the production of Turnbull's Blue.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

The mechanism of transmembrane delta muH+ generation in mitochondria by cytochrome c oxidase.

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.     

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.     

A simultaneous electrochemical impedance and quartz crystal microbalance study on antihuman immunoglobulin G adsorption and human immunoglobulin G reaction

The quartz crystal microbalance (QCM), in combination with electrochemical impedance spectroscopy (EIS), has been utilized to monitor in situ antihuman IgG (hIgG) adsorption on bare poly(o-phenylenediamine) (PPD)- and 1-dodecanethiol (C12SH)-modified Au electrodes and succeeding human IgG reaction, respectively. The resonant frequency (f) and the motional resistance (R(1)) of the piezoelectric quartz crystal (PQC) as well as electrochemical impedance (EI) parameters were measured and discussed. The standard heterogeneous rate constants of the ferricyanide/ferrocyanide couple before and after the antibody adsorption and antibody-antigen reactions were determined. The results show that the amount for antibody adsorption was the greatest on the most hydrophobic (1-dodecanethiol-modified) surface, while the antibody bioactivity was almost identical on the three kinds of surfaces. Two parameters simultaneously obtained, Deltaf and DeltaC(s) (interfacial capacitance), have been used for the first time to estimate both the association constant of the immunoreaction and the valence of antigen with satisfactory results. The proposed method may find wide application in interfacial biochemistry studies for its advantages in providing real-time multidimensional piezoelectric and electrochemical impedance information.     

A possible relationship between the effect of factors on photoactivation of photosystem II depleted of functional Mn and cytochrome b559

The photoassembly of the Mn₄CaO₅ cluster in Mn-depleted photosystem II preparations (photoactivation) was studied under the influence of oxidants, reductants and pH. New data on the effect of these factors on the photoactivation yield are presented. The presence of the oxidant, ferricyanide, negatively affected the photoactivation yield over the entire concentration range studied (0–1 mM). In contrast to ferricyanide, the addition of the reductant, ferrocyanide, up to 1 mM resulted in an increase in the photoactivation yield. Other reductants either did not significantly affect (diphenylcarbazide) or suppressed (ascorbate) the photoactivation yield. The effect of ferrocyanide on photoactivation were found to be similar dichlorophenolindophenol. Investigation of the photoactivation yield as a function of pH revealed that the maximum yield was observed at pH 6.5 in the presence of ferrocyanide and DCPIP, and at pH 5.5 without additives. In addition, the photoactivation yield at pH 5.5 was the same without and with the addition of ferrocyanide or dichlorophenolindophenol. Although ferricyanide suppressed the photoactivation, the photoactivation yield increased in the presence of ferricyanide by shifting the pH to the acidic region. The samples contained approximately 25 % of the HP cyt b₅₅₉, which was in the reduced state, as the absorbance at 559 nm was decreased upon addition of ferricyanide and subsequent addition of ferrocyanide returned the spectrum to the baseline. A possible relationship between the effect of factors on the photoactivation and the involvement of cyt b₅₅₉ in the protection of PSII from oxidative damage on the donor side is discussed.     

Thiamine: a mechanistic model of interaction with protein

Mechanistic model of thiamine-binding protein functioning which is based on the potential role of prototropic groups and hydrophobic environment around 5-beta-hydroxyethyl substituent of ligand has been proposed. As a model the chemical transformations of thiamine and its structural O-acyl substituted analogues in the presence of ferricyanide and phosphatic buffer in pH range 7,2-7,8 were investigated. The oxidation to the thiochrome and thiochrome derivatives is first order in substrate and ferricyanide concentrations. It is found that the reciprocal of the pseudo-first-order rate constant increases in ferrocyanide concentration at the constant oxidant concentration. Rate constants and partition ratios for reaction of thiamine, O-benzoylthiamine, O-(4-nitrobenzoyl)thiamine, O-(2-norbornoyl) thiamine, O-(1-norbornoyl)thiamine, O-(1-adamantoyl) thiamine, O-(2-adamantoyl) thiamine, O-(5-methyl-1-adamantyl)acetylthiamine, O-(2-adamantyl)acetylthiamine, O-(1-adamantyl)acetylthiamine were determined. The acceleration effect of hydrophobic fragment of O-acyl substituent is attributed to the formation of neutral tricyclic form in the step followed by electron transfer to ferricyanide. Mechanistic implications for possible transformation of thiamine in neutral tricyclic form at interaction with thiamine-binding protein are discussed.     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.     

A direct-colouring, metal precipitation method for the demonstration of arylsulphatases A and B

Summary A new, direct-colouring, metal precipitation method for the light microscopical demonstration of arylsulphatases A and B is described. It is based on the reducing capacity of nitrocatechol liberated by arylsulphatases fromp-nitrocatechol sulphate. The reaction is carried out in Karnovsky-Roots' copper ferricyanide incubation mixture at pH 5.0 or 5.5; the nitrocatechol liberated reduces ferricyanide to ferrocyanide, which in turn forms a brown precipitate with copper that indicates enzyme activity. Enzyme activity was localized in cytoplasmic particles compatible with a lysosomal localization of the enzyme. The histochemical reaction was inhibited by phosphate, which has been shown to inhibit arylsulphatases A and B in biochemical determinations. The method described is technically simple, and sections can be mounted in synthetic resin after dehydration.