Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of several mono- and dihydroxybenzene derivatives of various depigmenting potencies with L-3,4-dihydroxyphenylalanine-melanin

Certain mono- and dihydroxybenzene derivatives cause depigmentation of skin and hair, and appear to be selectively cytotoxic for melanized pigment cells. As direct physical and/or chemical interaction between depigmenter (DP) and pigment melanin may play a role in depigmentation, we have carried out preliminary studies in model systems where such interactions may easily be separated from effects due to tyrosinase, melanosomal proteins, and other components. We have used synthetic L-3,4-hydroxyphenylalanine (L-DOPA)-melanin as a protein-free model pigment and potassium ferricyanide as a model electron acceptor. Compounds studied were catechol, 4-t-butylcatechol, 4-methylcatechol, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, hydroquinone, 4-methoxyphenol, 4-t-butylphenol, and 2,6, di-t-butyl-4-methylphenol (BHT) in 0.1 M phosphate buffer, pH 7.4. These compounds vary widely in their ability to depigment hair and skin. Ferricyanide reduction by DP in the presence and absence of melanin was monitored spectrophotometrically. The sparingly soluble BHT and 4-t-butylphenol did not reduce ferricyanide in the absence or presence of melanin. For the other compounds, kinetic analysis demonstrated direct interaction between each DP and melanin. Except for dihydroxyphenylacetic acid, reduction kinetics were consistent with a mechanism involving noninteractive binding of both DP and ferricyanide to melanin prior to coupled electron transfer through the melanin backbone. Kinetic analysis afforded KB, a thermodynamic constant (M-1) for DP-melanin binding, and k', a rate parameter (M s-1) for electron transfer. A dimensionless enhancement factor (EF) was defined as k'KB/ks, with ks a pseudo-first-order constant (s-1) for ferricyanide reduction in the absence of melanin. Depending on the reductant, melanin either retards (EF less than 1) or accelerates (EF greater than 1) the rate of ferricyanide reduction. There appears to be a direct relationship between EF and depigmenting potency. There is no relationship between depigmenting power and the ability per se of the DP to bind to melanin or to reduce ferricyanide.     

Redox activity at the surface of oat root cells

Electron transport activity at the cell surface of intact oat seedlings (Avena sativa L. cv Garry) was examined by measuring the oxidation and/or reduction of agents in the medium bathing the roots. Oxidation of NADH with or without added electron acceptors and reduction of ferricyanide by an endogenous electron donor were detected. The activities appear to be due to electron transfer at, or across, the plasma membrane and not due to reagent uptake or leakage of oxidants or reductants. NADH-ferricyanide oxidoreductase activity was also detected in plasma membrane-enriched preparations from Avena roots. Based on redox responses to pH, various ions, and to a variety of electron donors and acceptors, the results indicate that more than one electron transport system is present at the plasma membrane.     

Electron transfer properties of melanin.

Melanin synthesized from mushroom tyrosinase and 3,4-dihydroxyphenylalanine has been shown to oxidize NADH and NADPH, reduce ferricyanide, oxidized forms of cytochrome c and dichlorophenolindophenol, and catalyze the coupled oxidation of NADH and reduction of ferricyanide. Kinetic studies involving the determination of initial velocity at various concentrations of substrates and product inhibition measurements have been carried out on the NADH-ferricyanide-melanin reaction. The results are consistent with a ping-pong mechanism in which one product is formed prior to the reaction of melanin with the second substrate involving the reversible oxidation and reduction of melanin during the reaction. It may be concluded that melanin is capable of acting as an electron transfer agent in several reduction-oxidation systems.     

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes

An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., alpha-lipoic acid (ALA), 5,5'-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl- from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

Kinetic and inhibition studies on reduction of diphenyl sulfoxide by guinea pig liver aldehyde oxidase

To characterize the properties of diphenyl sulfoxide (DPSO) as a new type of electron acceptor for guinea pig liver aldehyde oxidase (AO), we compared the kinetics of the reductions of DPSO and other classical electron acceptors such as O2 and ferricyanide. The double-reciprocal plot of the 2-hydroxypyrimidine (2-OH PM)-linked DPSO reduction with the highly purified enzyme was biphasic. Similar biphasic plots were obtained with the reductions of other electron acceptors. Only the lower Km value, which was obtained by extrapolation of the plot at lower concentrations of 2-OH PM, was identical with that shown by the freshly prepared crude enzyme. DPSO as well as menadione progressively inhibited the reductions of O2 and ferricyanide with time. Menadione inhibited the DPSO reduction noncompetitively with respect to 2-OH PM and competitively with respect to DPSO, while its mode of inhibition of ferricyanide reduction was uncompetitive for either the electron donor or the acceptor. On the other hand, DPSO showed an uncompetitive and a noncompetitive inhibition of ferricyanide reduction with respect to 2-OH PM and ferricyanide, respectively. These results may indicate that DPSO interacts with the enzyme at the same site as menadione, and thereby when other electron acceptors are present it serves as an actual inhibitor rather than as an electron acceptor for AO.     

Activation of electron transport at the terminal site of the respiratory chain by the submitochondrial fluid from the rat and guinea pig liver

Supermitochondrial liquid (SL) of rat and guinea-pig liver increases the activity of 2, 3, 5 triphenyltetrasolium chloride (TPC) and tetrasolium violet (TV) reduction at succinate, NADH and NADPH oxidation by mitochondria (MC). SL contains an activating factor A, being evidently of a protein nature and factor B, increasing the activating activity of factor A. NAD, NADP, NADH and NADP at 5 x 10(-5)-1 x 10(-4) M concentration activate the TPC and TV reduction at succinate oxidation by mitochondria. TPC and TV reduction at succinate and NADP oxidation by mitochondria makes antimicin and cyanide sensitive. SL does not influence succinate dehydrogenase activity, when used as electron acceptors of ferricyanide, blue Vurster, cytochrome C, blue and violet nitrotetrasolium. Activation of electron transfer chair between cytochrome C and oxygen is supposed to be responsible for such an effect.     

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Iron stress-induced redox reactions in bean roots

Iron stress-induced and constitutive redox activity of bean ( Phaseolus vulgaris L. cv. Delinel) roots was measured on intact plants using FeEDTA and ferricyanide as electron acceptors. The presence of the translation inhibitor cycloheximide caused a decrease in the reduction of both oxidants. However, a differential decline in the reduction rates of FeEDTA and ferricyanide was observed, suggesting enzyme heterogeneity. In the presence of the H⁺ -ATPase inhibitor vanadate, the reduction of FeEDTA was nearly completely suppressed in both Fe-deficient (–Fe) and Fe-sufficient (+Fe) plants, providing evidence for an involvement of plasma membrane-bound ATPase activity in the regulation of the reduction process. The inhibition of the ferricyanide reduction by vanadate was restricted to –Fe plants.
The data are interpreted in terms of simultaneous operation of distinct redox systems in roots of iron-deficient bean plants. The role of proton extrusion in iron stress-induced electron transfer is discussed.     

Functional intermediates in the reaction of membrane-bound cytochrome oxidase with oxygen.

Flash photolysis of the membrane-bound cytochrome oxidase/carbon monoxide compound in the presence of oxygen at low temperatures and in the frozen state leads to the formation of three types of intermediates functional in electron transfer in cytochrome oxidase and reduction of oxygen by cytochrome oxidase. The first category (A) does not involve electron transfer to oxygen between -125 degrees and -105 degrees, and includes oxy compounds which are spectroscopically similar for the completely reduced oxidase (Cu1+alpha3(2+)-O2) or for the ferricyanide-pretreated oxidase (Cu2+alpha3(3+)-O2). Oxygen is readily dissociated from compounds of type A. The second category (B) involves oxidation of the heme and the copper moiety of the reduced oxidase to form a peroxy compound (Cu2+alpha 3(3+)-O2=or Cu2+alpha3(2+)-O2H2) in the temperature range from -105 degrees to -60 degrees. Above -60 degrees, compounds of type B serve as effective electron acceptors from cytochromes a, c, and c1. The third category (C) is formed above -100 degrees from mixed valency states of the oxidase obtained by ferricyanide pretreatment, and may involve higher valency states of the heme iron (Cu2+alpha3(4+)-O2=). These compounds act as electron acceptors for the respiratory chain and as functional intermediates in oxygen reduction. The remarkable features of cytochrome oxidase are its highly dissociable "oxy" compound and its extremely effective electron donor reaction which converts this rapidly to tightly bound reduced oxygen and oxidized oxidase.     

Transmembrane electron transfer in an enzyme-phospholipid complex

Purified malate-vitamin K reductase (MKR) from $\text{Mycobacterium phlei}$, which requires phospholipid and FAD for the enzymatic reduction of dye electron acceptors, was used as an electron transfer system in combination with synthetic phospholipid vesicles. Ferricyanide entrapped inside the sonically prepared vesicles was enzymatically reduced by MKR. The rate of reduction of ferricyanide by MKR was dependent upon a lipid soluble mediator. The rate of enzymatic reduction of ferricyanide by malate-vitamin K reductase was increased by the addition of phenazine methosulfate (PMS) or benzoquinone.     

Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.     

Humics as an electron donor for anaerobic respiration

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHQDS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens , Geobacter sulphurreducens , Geothrix fermentans , Shewanella alga , Wolinella succinogenes and ' S. barnesii '. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans , could not. AHQDS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes . Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.     

Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein.

The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported. Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants. (2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes. (4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction. Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants.     

Impermeant electron acceptors and donors to the plasma membrane-bound respiratory chain of intact cyanobacterium Anacystis nidulans

Membrane-impermeant redox compounds ferricyanide and horse heart ferrocytochrome c acted as electron acceptor and donor, respectively, for intact cells or spheroplasts of Anacystis nidulans (Synechococcus ATCC 27144) in the dark. The anaerobic reduction of ferricyanide was faster than aerobic reduction. KCN significantly enhanced the reaction under aerobic conditions. Light did not influence ferricyanide reduction. The oxidation of exogenous ferrocytochrome c was oxygen-dependent and inhibited by KCN. Either type of redox reaction was accompanied by vectorial proton translocation out of the cells. Arrhenius plots for the temperature dependence of both ferricyanide reduction and cytochrome c oxidation gave one distinct break point reflecting the lipid phase transition temperature of the plasma membrane. The results are presented as evidence for a respiratory chain in the plasma membrane of A. nidulans.     

Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

The chromaffin vesicle of the adrenal medulla contains a transmembrane electron carrier that may provide reducing equivalents for dopamine beta-hydroxylase in vivo. This electron-transfer system can be assayed by trapping ascorbic acid inside resealed membrane vesicles (ghosts), adding an external electron acceptor such as ferricytochrome c or ferricyanide, and following the reduction of these acceptors spectrophotometrically. Cytochrome c reduction is more rapid at high pH and is proportional to the amount of chromaffin-vesicle ghosts, at least at low ghost concentrations. At pH 7.0, ghosts loaded with 100 mM ascorbic acid reduce 60 microM cytochrome c at a rate of 0.035 +/- 0.010 mu equiv min-1 (mg of protein)-1 and 200 microM ferricyanide at a rate of 2.3 +/- 0.3 mu equiv min-1 (mg of protein)-1. The rate of cytochrome c reduction is accelerated to 0.105 +/- 0.021 mu equiv min-1 (mg of protein)-1 when cytochrome c is pretreated with equimolar ferrocyanide. Pretreatment of cytochrome c with ferricyanide also causes a rapid rate of reduction, but only after an initial delay. The ferrocyanide-stimulated rate of cytochrome c reduction is further accelerated by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), probably because FCCP dissipates the membrane potential generated by electron transfer. These rates of electron transfer are sufficient to account for electron transfer to dopamine beta-hydroxylase in vivo and are consistent with the mediation of electron transfer by cytochrome b-561.     

Selective thiol inhibition of ferricyanide reduction in photosystem II of spinach chloroplasts

Selective inhibition of ferricyanide reduction in photosystem II by lipophilic thiols indicates a unique pathway of electron transport, which is not involved in reduction of class III acceptors or transfer of electrons to photosystem I. Both aromatic and aliphatic thiols induce the inhibition, but thiol binding reagents such as p-hydroxymercuribenzoate or N-ethylmaleimide do not inhibit. The inhibition can be observed using either dibromothymoquinone or bathophenanthroline to direct electrons away from photosystem I. No pretreatment of chloroplasts with thiols in the light was necessary to inhibit ferricyanide reduction by photosystem II or the O₂ evolution associated with ferricyanide reduction.