一种改进的乳酸脱氢酶同工酶染色法

以N-甲基吩嗪甲基硫酸盐（PMS）为介体,使乳酸脱氢酶（LDH）同工酶催化反应产生的NADH将铁氰化钾还原为亚铁氰化钾,利用亚铁氰化钾与三氯化铁反应生成普鲁士蓝的性质,实现对乳酸脱氢酶同工酶进行染色的目的.     

Glycogen, its chemistry and morphological appearance in the electron microscope. III. Identification of the tissue ligands involved in the glycogen contrast staining reaction with the osmium (VI)-iron(II) complex

Synopsis By application of appropriate blocking reactions (acetylation, de-amination, methylation and NaHSO₃-treatment) it is demonstrated that the tissue ligands involved in the selective glycogen contrast staining reaction with the Os^VI. Fe^II complex (known to be present in the combination K₂OsO ₄ ^K ₄Fe(CN)₆) are the glycogen C₂–C₃ di-hydroxyl groups. Deliberate conversion of the diols into di-aldehydes and (di-)carboxyl groups by the application of specific oxidative agents followed, by application of the Os^VI.Fe^II-complex results morphologically in identical selective contrast staining of glycogen.By applying appropriate blocking reactions to such pre-oxidized aldehyde fixed glycogen, evidence is accumulated that K₂OsO₄ and K₃Fe(CN)₆ are unable to oxidize diols, whereas OsO₄ and H₂O₂ are able to convert diols into carboxyl groups.From these results it is concluded that in the combination K₂OsO ₄ ^K ₄Fe(CN)₆ the Os^VI.Fe^II complex reacts with unchanged diols in the glycogen, whereas the OsO₄ in the combination OsO ₄ ^K ₄Fe(CN)₆ can petentially create carboxyl groups in the aldehydefixed glycogen.The addition of urea to the two glycogen contrasting combinations (K₂OsO ₄ ^K ₄Fe(CN)₆ or OsO ₄ ^K ₄Fe(CN)₆), also emphasizes that, although morphologically both combinations produceidentical contrast stained glycogen, chemically the contrast staining is apparently obtained in a different way, as urea prevented the contrast for mation in the glycogen by the combination K₂OsO ₄ ^K ₄Fe(CN)₆, but not by the combination OsO ₄ ^K ₃Fe(CN)₆.     

Protein control of the formation and decomposition of the CYP119 and CYP101 aryl-iron complexes

CYP119, the first thermophilic P450 enzyme, reacts much more slowly than CYP101 (P450cam) with aryldiazenes to give σ-bonded aryl-iron complexes. The CYP119 complexes are stable anaerobically at 80 °C but are readily oxidized by O₂ to give the N-arylprotoporphyrin IX regioisomers. The aryl shift can also be initiated in the absence of O₂ by K₃Fe(CN)₆. In contrast, the corresponding CYP101 complexes are insensitive to O₂ but decompose at temperatures above 50 °C owing to denaturation of the protein. The rate of the CYP119 aryl shift is decreased by electron-withdrawing substituents, with ρ=−1.50 for both the O₂- and K₃Fe(CN)₆-dependent reactions. A similar dependence (ρ=−0.90) is observed for the K₃Fe(CN)₆-dependent CYP101 shift. The enthalpies and entropies of activation suggest that the CYP119 and CYP101 K₃Fe(CN)₆-mediated reactions are similar, but the CYP119 O₂-dependent reaction proceeds via a different transition state. In all cases, the rate-determining step is oxidation of the aryl-iron complex. The temperature dependence of the O₂- and K₃Fe(CN)₆-dependent CYP119 shifts provides evidence for temperature-dependent equilibration of two active site conformations. The oxygen sensitivity of the CYP119 aryl-iron complexes, and the temperature dependence of their rearragement, reflect the unique active site properties of this thermophilic P450 enzyme. Received: 12 August 1999 / Accepted: 8 December 1999     

TEMPERATURE ACTIVATION OF THE UREASE-UREA SYSTEM USING CRUDE AND CRYSTALLINE UREASE

1. The hydrolysis of urea catalyzed by jack bean meal has been followed by determining colorimetrically after Nesslerization the ammonia nitrogen, and volumetrically the carbon dioxide liberated at successive intervals during the reaction. During the early part of hydrolysis the rate of ammonia or carbon dioxide liberation is constant for all the urease solutions which were used. 2. When log rate of NH₃ or CO₂ formation was plotted against 1/T, the points fell along a straight line, the slope of which corresponded to an activation energy of either 8,700 or 11,700 calories per gram mol. Frequently urease, when dissolved in sulfite solution, was characterized by an activation energy of 11,700 below and 8,700 above the critical temperature of about 23°C. At high temperatures the plotted points fell off from the curve due to temperature inactivation. 3. Essentially the same results on temperature activation were obtained with crude jack bean meal, Arlco urease, crystalline urease not recrystallized, and crystalline urease once recrystallized. The temperature characteristic which was obtained depended in part upon the composition of the medium. When dissolved in water, or aqueous solutions of glycerine, KCN, Na₂S₂O₂, cystine, Na₂SO₄, and K₄Fe(CN)₆, the temperature characteristic or µ of urease is 8,700. On the other hand, when urease is dissolved in solutions of K₃Fe(CN)₆ or H₂O₂ the µ value is 11,700. When dissolved in a solution containing Na₂SO₃ and NaHSO₃ the µ value may be either 8,700 or 11,700 over the whole temperature range, or 11,700 below and 8,700 above 23°C. 4. When crystalline urease is dissolved in varying mixtures of K₄Fe(CN)₆ and K₃Fe(CN)₆, the temperature characteristic depends upon the oxidation-reduction potential of the digest. When E_h is greater than +0.46 volt µ = 11,700, when less than +0.42 volt µ = 8,700, when between +0.42 – +0.46 µ = 11,700 below and 8,700 above the critical temperature. 5. It is suggested that in reducing or in indifferent solutions the configuration of the urease molecule (as determined especially by SH groups present) is such that the activation energy is 8,700 calories. In oxidizing solutions the urease molecule has been so altered (perhaps by the oxidation of the SH groups) as to be partly inactivated and now has an activation energy of 11,700. Such changes in the urease molecule are reversible (unless oxidation has proceeded too far) and are accompanied by a corresponding change in the activation energy.     

The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation

These studies were designed to compare the effects of nitric oxide (NO) generating compounds with those of several iron containing, compounds which do not generate NO on glutamate receptor function. Stimulation of primary cultures of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainate results in the elevation of intracellular calcium ([Ca²⁺]_i) and cGMP and the release of glutamate. The iron containing compounds, sodium nitroprusside (SNP), potassium ferrocyanide (K₄Fe(CN)₆) and potassium ferricyanide (K₃Fe(CN)₆) decrease the NMDA-induced release of glutamate. SNP is the only compound of the above 3 agents which generates NO. A non-iron, NO generating compound, S-nitroso-N-acetylpenicillamin (SNAP), has no effect on the NMDA-induced glutamate release. Potassium ferrocyanide (Fe II), but not potassium ferricyanide (Fe III), blocks NMDA-induced cGMP elevations after 3 min exposure times. This contrasts with the NO generating compounds (both SNP and SNAP) which elevate cGMP levels. Furthermore, both potassium ferrocyanide (Fe II) and SNP (Fe II) suppress the elevation of [Ca²⁺]_i induced by NMDA but neither potassium ferricyanide (Fe III) nor SNAP are effective in this regard. These effects are also independent of cyanide as another Fe II compound, ferrous sulfate (FeSO₄) is also able to suppress NMDA-induced elevations of [Ca²⁺]_i SNP was unable to suppress kainate receptor functions. Collectively, these results indicate that Fe II, independently of NO, has effects on NMDA receptor function.     

Synthesis, magnetic properties and crystal structures of two compounds: [Cu(en)(H2O)]2[Fe(CN)6]·4H2O and [Cu(en)2][KFe(CN)6] (en=ethylenediamine)

Two new heterometallic complexes, [Cu(en)(H₂O)]₂[Fe(CN)₆]·4H₂O (1) and [Cu(en)₂][KFe(CN)₆] (2), have been isolated from the reactions of CuCl₂ and en with K₃[Fe(CN)₆] in different molar ratios. Both complexes have been characterized by X-ray analyses, IR spectra and elemental analyses. Complex 1 is a cyanide bridged bimetallic assembly, its crystal structure consists of a two-dimensional polymeric sheet with two different rings, one a four-membered square ring and another a 12-membered hexagonal ring. The Fe(II) ion of 1 has two terminal, two linear bridging and two 1,1 en-on bridging cyanide groups. In the crystal structure of 2, the neighboring [Fe(CN)₆]³⁻ units are bridged by the K⁺ and the [K[Fe(CN)₆]]²⁻ units forming a three-dimensional network structure. The [Cu(en)₂]²⁺ units fill in the holes of the network acting as counter cations and charge compensations. Variable temperature magnetic susceptibility studies of 1 indicate that the complex exhibits ferromagnetic interaction between the Cu(II) ions.     

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe³⁺ ions.Erythrocytes were exposed to various concentrations of CuSO₄, Fe(NO₃)₃, and K₃Fe(CN)₆ for up to 5 h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe²⁺ to Cu²⁺. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO₃)₃. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO₄ increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 μM. This effect was decreased by potassium ferricyanide and Fe(NO₃)₃ only after 1 and 3 h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 μM CuSO₄ as well as 20 μM Fe(NO₃)₃ and 10 mM K₃Fe(CN)₆ induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K₃Fe(CN)₆. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO₃)₃.     

Chemical evolution of iron containing enzymes: Mixed ligand complexes of iron as intermediary steps

Activities of the iron complexes of evolutionary importance like K₄[Fe(CN)₆], K₄[Fe(CN)₅(gly)], and K₄[Fe(CN)₅(trigly)] have been tested towards some redox reactions of biological significance, namely, decomposition of hydrogen peroxide, dehydrogenation of NADH and ascorbic acid both coupled with reduction of methylene blue. It has been observed that the catalytic activities of iron (II) complexes towards the redox reactions studied at pH 9.18 followed the order, K₄[Fe(CN)₆]4[Fe(CN)₅(gly)]4[Fe(CN)₅(trigly)]. Decomposition of H₂O₂ catalysed by cyanocomplexes of iron (II) has been discussed through the formation of an innersphere complex in which loosly bound ligands like, glycine and triglycine are replaced by hydroperoxide ion. A tentative mechanism for the catalysed decomposition of H₂O₂ has been discussed.Based upon the experimental observations a hypothesis on the evolution of iron containing enzymes has been envisaged as: iron(II) ion iron(II) cyanide complexes mixed ligand iron(II) cyanide and amino acid complexes iron(II) complexes of macromolecules proenzyme or early enzyme containing iron(II).     

Ultrastructural chemical reaction to detect saturated phospholipids of a natural surfactant from pig lungs

The ultrastructure of the natural surfactant from pig lungs fixed by the tricomplex method with Pb(NO₃)₂ and K₃Fe(CN)₆ reveals its lamellar phase (ordered configuration). The lamellar forms are usually associated with being surface active.     

A plasmamembrane redox system and proton transport in isolated mesophyll cells

Potassium ferricyanide (K₃Fe[CN]₆) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competent Asparagus sprengeri Regel mesophyll cells. Rates of Fe(CN)₆³⁻ reduction and H⁺ efflux were measured with or without illumination. On the addition of 1 millimolar Fe(CN)₆³⁻ to nonilluminated cell suspensions acidification of the medium indicated an H⁺ efflux of 1.54 nanomoles H⁺/10⁶ cells per minute. Simultaneous Fe(CN)₆³⁻ reduction occurred at a rate of 1.55 nanomoles Fe(CN)₆³⁻/10⁶ cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H⁺/10⁶ cells per minute and 22.9 nanomoles Fe(CN)₆³⁻/10⁶ cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H⁺ efflux in illuminated or nonilluminated cells but had no influence on Fe(CN)₆³⁻ dependent H⁺ efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H⁺. The present report extends these observations to photosynthetically competent mesophyll cells. The results indicate a transport process independent of ATP driven H⁺ efflux which operates with a H⁺/e⁻ stoichiometry of one.     

Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I

Crystals of Azotobacter vinelandii ferredoxin I (FdI) have been soaked in solutions containing K₃Fe(CN)₆ in order to study the oxidation of the [3Fe-4S] and [4Fe-4S] clusters in the protein. Ferricyanide treatment results in partial loss of Fe and S from each cluster accompanied by alteration of Fe-S bonds. The effects of oxidation can be quantitated by crystallographic refinement when each [Fe-S] cluster is modeled as having a single, average structure with non-standard geometry. The oxidized clusters refined at 2.1-Å resolution display statistically significant deviations from geometric ideality. If interpreted in terms of atomic shifts these deviations indicate that each cluster first loses an inorganic S atom. In each case an Fe atom bonded to this S separates from the remaining atoms of the cluster such that the [3Fe-4S] and [4Fe-4S] clusters partially decompose into a single Fe plus 2Fe and 3Fe fragments. The extent of structural changes observed are essentially the same in crystals soaked at 3?:?1, 9?:?1 and 30?:?1 mole ratio of K₃ Fe(CN)₆?:?FdI, suggesting that the crystal lattice permits limited oxidation reactions to occur at a low mole ratio but restricts conformational changes from occurring that may be required for more extensive oxidative reactions at higher mole ratio. The results are relevant to understanding the transformations which may take place when [Fe-S] proteins are deliberately oxidized with ferricyanide.     

Specific cation effect on quenching reactions of excited tris(α,α′-diimine) ruthenium(II) and chromium(III) complexes by cyanide complexes in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(NN)₃]²⁺ (NN=2,2′-bipyridine(bpy), 1,10-phenanthrorine(phen)) and [Cr(bpy)₃]³⁺ by [Cr(CN)₆]³⁻, [Fe(CN)₆]³⁻ and [Ni(CN)₄]²⁻ in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The quenching rate constants in [Ru(NN)₃]²⁺-[Cr(CN)₆]³⁻ and [Cr(bpy)₃]³⁺-[Cr(CN)₆]³⁻ systems are changed by the cations as Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺. On the other hand, the rate constants in [Ru(NN)₃]²⁺-[Fe(CN)₆]³⁻ and [Ru(NN)₃]²⁺-[Ni(CN)₄]²⁻ systems, which are diffusion-controlled reactions, are not varied by the alkali metal cations. The obtained order (Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺) of the quenching rate constant is quite different from salt effects, Li⁺<Na⁺<K⁺<Rb⁺<Cs⁺, which have been obtained in the electron transfer reactions between complex anions.     

Kinetics and mechanism of the reaction between 1,2-diaminopropanetetra-acetatoferrate(III) and cyanide ion

The present study examines the kinetics and mechanism of the system [FePDTA(OH)]²⁻ + 5CN⁻ ⇌ [Fe(CN)₅OH]³⁻ + PDTA⁴⁻ at pH= 11.0±0.02, I= 0.25 M and temperature = 25 ± 0.1 °C. The reaction has been studied spectrophotometrically at 395 nm (λ_max of [Fe(CN)₅OH]³⁻). The data show that the reaction has three distinguishable stages; the first stage is formation of [Fe(CN)₅OH]³⁻, the second is conversion of [Fe(CN)₅OH)]³⁻ to [Fe(CN)₆]³⁻ and last is reduction of [Fe(CN)₆]³⁻ to [Fe(CN)₆]³⁻ by the released ligand, viz., PDTA. The first reaction shows variable order dependence on cyanide concentration, one at high cyanide concentration and two at low cyanide concentration. The second reaction exhibits first order dependence on the concentration of [Fe(CN)₅OH]³⁻ as well as cyanide. The reverse reaction between [Fe(CN)₅OH]³⁻ and PDTA is first order in [Fe(CN)₅OH]³⁻ and PDTA, and inverse first order in cyanide. On the basis of forward and reverse rate studies, a five-step mechanism has been proposed for the first reaction.     

Preferential uptake of ammonium ions by zinc ferrocyanide

The concentration of ammonia from dilute aqueous solution could have facilitated many prebiotic reactions. This may be especially true if this concentration involves incorporation into an organized medium. We have shown that (unlike iron(III) ferrocy anide) zinc ferrocyanide, Zn₂ Fe (CN)₆·xH₂O, preferentially takes up ammonium ions from 0.01 M NH₄Cl to give the known material Zn₃(NH₄)₂ [Fe(CN)₆]₂·xH₂O, even in the presence of 0.01M KC1. KC1 alone gave Zn₃K₂[Fe(CN)₆]₂·xH₂O. Products were characterized by elemental (CHN) analysis and powder X-ray diffraction (XRD). We attribute the remarkable specificity for the ammonium ion to the open framework of the product, which offers enough space for hydrogen-bonded ammonium ions, and infer that other inorganic materials with internal spaces rich in water may show a similar preference.     

Flowering behavior of the hybrids between strains 6746 and 371 of Lemna paucicostata hegelm

Lemna paucicostata Hegelm. 6746, a short-day plant, flowers even under continuous light when CuSo₄, AgNO₃, K₃[Fe(CN)₆], or benzoic acid are added to the medium, or when blue light is given exclusively. All other strains tested, including strain 371, did not flower under any of these conditions. Crossing between strains 371 (♀) and 6746 (♂) produced some seeds, although the reciprocal crossing did not. None of the hybrids flowered in response to AgNO₃, or blue light, and only a few flowered in response to CuSO₄. Most of the hybrids, on the other hand, flowered in response to K₃[Fe(CN)₆] or benzoic acid, although the responses were lower than the mean of the parental. Further analysis could not be made because self-pollination or crossing between the hybrids did not produce any seeds. However, since sensitivities to the above 5 factors were passed on to the F₁ hybrids in varying degrees, the extraordinary high sensitivities to these factors of strain 6746 are considered to be governed by different genes. Otherwise, a single (or a few) gene(s) governing the sensitivities to these factors would be differently expressed in the hybrids.     

Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Effects of photoinhibition on the PS II acceptor side including the endogenous high spin Fe2+ in thylakoids,PS II-membrane fragments and PS II core complexes

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 M K₃[Fe(CN)₆]; whereas, a characteristic binary oscillation was observed in the presence of 100 M phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K₃[Fe(CN)₆] for only 5 min with white light (180 W m^-2) largely eliminates the very fast fluorescence decay after the first flash due to Q_A ^- reoxidation by preoxidized endogenous non-heme Fe³⁺, while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6–9 min) and the slow degradation component (60–75 min) are practically independent of the absence or presence of K₃[Fe(CN)₆] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe²⁺ located between Q_A and Q_B is shown to become highly susceptible to modification by photoinhibition in the presence of K₃[Fe(CN)₆] (and other exogenous acceptors), while the rate constant of Q_A ^- reoxidation by Q_B(Q_B ^-) and other acceptors (except the special reaction via Fe³⁺) is markedly less affected by a short photoinhibition. The equilibrium constant between Q_A ^- and Q_B(Q_B ^-) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.     

Photoinhibition as a function of the ambient redox potential in Tris-washed PS II membrane fragments

The mode of photoinhibition as a function of the ambient redox potential (E_ambient) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, ΔA^total ₈₃₀, as a measure of the capacity to form the `stable' radical pair, P680^+ċ Q^−ċ _A, drastically decreases during a 10 min photoinhibition at E_ambient values below +350 mV; (b) conversely, the normalized extent of the 18 μs relaxation kinetics, ΔA^{18 μ s} ₈₃₀ as a measure of the electron transfer from Y_Z to P680^+ċ becomes highly susceptible to light stress when E_ambient exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K₃[Fe(CN)₆] and K₄[Fe(CN)₆] in the suspension during photoinhibition but rather depends on the E_m-values; (e) qualitatively similar features are observed when the redox buffer system K₃[Fe(CN)₆]/Na₂S₂O₄ is replaced by K₂[IrCl₆]/Na₂S₂O₄; (f) the characteristic E_ambient-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559. This revised version was published online in June 2006 with corrections to the Cover Date.