Inhibition Of Ferricyanide Reduction Research Articles

The NADH oxidation domain of Complex I: do bacterial and mitochondrial enzymes catalyze ferricyanide reduction similarly?

The hexammineruthenium (HAR) and ferricyanide reductase activities of Complex I (H +-translocating NADH:ubiquinone reductase) from Paracoccus denitrificans and bovine heart mitochondria were studied. The rates of HAR reduction are high, and its steady-state kinetics is similar in both P. denitrificans and bovine Complex I. The deamino-NADH:HAR reductase activity of Complex I from both sources is significantly higher than the respective activity in the presence of NADH. The HAR reductase activity of the bacterial and mitochondrial Complex I is similarly and strongly pH dependent. The p K a of this activity could not be determined, however, due to low stability of the enzymes at pH values above 8.0. In contrast to the high similarity between bovine and P. denitrificans Complex I as far as HAR reduction is concerned, the ferricyanide reductase activity of the bacterial enzyme is much lower than in mitochondria. Moreover, ferricyanide reduction in P. denitrificans, but not bovine mitochondria, is partially sensitive to dicyclohexylcarbodiimide (T. Yagi, Biochemistry 26 (1987) 2822–2828). On the other hand, the inhibition of ferricyanide reduction by high concentration of NADH, a typical phenomenon in bovine Complex I, is much weaker in the bacterial enzyme. The functional differences between the two enzymes might be linked to the properties of their binuclear Fe–S clusters.

Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

Cultured cells of Rosa damascena have been used as a model for studies of responses of plant cells to various stresses, including UV radiation, protein-synthesis inhibitors, and elicitors from pathogens. Many of the responses involve reactions at the plasma membrane: efflux of K+, changes in the acid balance between cytoplasm and external medium, synthesis of H2O2, and inhibition of ferricyanide reduction. In previous studies, the cells have typically been washed with a solution of low ionic strength. We now show that this washing procedure results in changes in the protein composition of the plasma membrane, in the labeling of the proteins in the plasma membrane, and in the specific activity of ATPase in purified plasma membrane vesicles. Also, compared to the unwashed cells, the washed cells show less net K+ efflux after UV-C and Phytophthora elicitor treatments; more synthesis of H2O2 after UV-C and a pattern of accumulation of H2O2 after elicitor treatment that shows a delayed but higher peak; and more inhibition of ferricyanide reduction after UV-C, but not after elicitor treatment. The results suggest that washing has differential effects on the mechanisms by which cultured plant cells perceive or respond to two stresses, UV-C and elicitor.

Phytophthora elicitor inhibits ferricyanide reduction by cultured rose cells

Suspension cultured cells of Rosa damascena Mill. cv. Gloire de Guilan exhibited normal plasma membrane electron transport activities, including the reduction of extracellular ferricyanide to ferrocyanide, the reduction of Fe(III) chelate, and the oxidation of exogenous NADH. Elicitor preparations from autoclaved cell walls of Phytophthora sp. or from clarified fungal growth medium inhibited ferricyanide reduction (and stimulated ferrocyanide oxidation) by the rose cells. The effect occurred after a lag period of 10–15 min and lasted for at least 60 min. There was no inhibition of reduction of Fe(III) chelate; there was a stimulation of oxidation of exogenous NADH. The effect was observed after a treatment of the cells with salicylate, but not after treatment with ultraviolet radiation or cycloheximide, agents that otherwise mimic the effect of elicitor on rose cell plasma membrane activities. There was no effect of elicitor added in vivo or in vitro on the NADH-ferricyanide oxidoreductase activity of isolated plasma membrane. The inhibition of ferricyanide reduction and oxidation of ferrocyanide could not be attributed to an elicitor-dependent accumulation of O − 2 or H 2O 2, since added superoxide dismutase and catalase did not block the inhibition of ferricyanide reduction. It is suggested that the inhibition of ferricyanide reduction was related to a change in the redox state of cytoplasmic NADH/NAD +.

Inhibition by plastoquinone analogues of ferricyanide reduction by a Photosystem II chloroplast preparation

There is wide agreement that inhibition of plastoquinone function by plastoquinone analogues, of which the best known is dibromothymoquinone (DBMIB), inhibits electron transport from Photosystem II (PS II) to Photosystem I (PS I), but does not inhibit electron transport that is dependent on PS II alone. In contrast, we have recently presented evidence in support of a hypothesis for a hitherto unrecognized additional role of plastoquinone in the conductance of protons derived from the photooxidation of water by PS II (Arnon, D.I. and Tang, G.M.-S. (1985) Biochim. Biophys. Acta 809, 167–172). Since the transport of water-derived electrons and protons is coupled, this hypothesis predicts that photosynthetic electron transport by PS II alone would still be sensitive to DBMIB inhibition. We now report that this prediction has been verified by obtaining inhibition by low concentrations of DBMIB of electron transport from water to ferricyanide in a PS II preparation depleted of plastocyanin and PS I. The preparation consisted of inside-out vesicles isolated by the aqueous polymer two-phase partition method. The pattern of DBMIB inhibition in the PS II preparation was the same as that in unfractionated chloroplasts: inhibition of ferricyanide reduction was relieved by the addition of catalytic amounts of one of several lipophilic acceptors that appear to bypass the site of DBMIB inhibition at the Rieske FeS center of the cytochrome f b 6 complex. The bearing of these findings on the role of plastoquinones in the conductance of protons released by the photooxidation of water is discussed.

Inhibition of ferricyanide reduction in chloroplast particles by anaerobicity

The reduction of ferricyanide by spinach and pea chloroplast particles was the same under aerobic or anaerobic conditions. The rate of ferricyanide reduction by these particles uncoupled by ammonium chloride or carboxycyanide p-trifluoromethoxyhydrazone, or Chlamydomonas reinhardi or Euglena gracilis particles uncoupled by sonic oscillation, was inhibited by anaerobic conditions. This inhibition by anaerobicity may explain the cessation of the photolysis of water as measured by H 2 production in reconstituted preparations consisting of chloroplast particles, ferredoxin and fully active hydrogenase.

Evidence for multiple sites of ferricyanide reduction in chloroplasts.

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.

Inhibition of Ferricyanide Reduction in Chloroplasts Prepared from Water‐stressed Cotton Leaves1

Water stress was applied to cotton (Gossypium hirsutum L.) chloroplasts and leaves by the following treatments: (i) prepared chloroplasts were. placed in assay media different osmotic potentials; (ii) cut shoots were allowed to transpire under illumination; (iii) whole potted plants were held for 5 days without irrigation; and (iv) cell sap was expressed from individual leaves via the xylem in a pressure bomb. These treatments caused a decrease in ferricyanide reduction when isolated chloroplasts were assayed potentiometrically for the Hill reaction. The above treatments caused (i) 1.7%, (ii) 1.4%, (iii) 1.8%, and (iv) 1.9% decrease of initial activity rcspectively per bar of water potential decline. This decrease in activity, which was nearly linear with the degree of stress, occurred from —3 to —30 bars leaf water potential in treatments ], 3, and 4, and from —15 to —30 bars in treatment 2. These results indicate that water stress in the leaf can directly influence the photochemical activity of the chloroplasts.