Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

Interaction of beta-lactoglobulin and cytochrome c: complex formation and iron reduction.

β-Lactoglobulin forms a soluble complex with cytochrome c in mildly alkaline solutions of low ionic strength. Sedimentation velocity experiments suggest that the complex (maximum s₂₀ = 3.7) consists of one cytochrome c molecule per β-lactoglobulin monomer unit. At pH 8 or higher, the presence of β-lactoglobulin causes reduction of ferri- to ferrocytochrome c. The initial rate of reduction at a single temperature depends primarily on the concentration of β-lactoglobulin, although the final percentage ferrocytochrome c obtained is constant at molar ratios of three or more β-lactoglobulin monomers to one cytochrome c molecule. The temperature dependence of the initial rate of iron reduction resembles that for alkaline denaturation of β-lactoglobulin. The displacement of N-dansylaziridine, a sulfhydryl specific dye, from bovine β-lactoglobulin during iron reduction, and the formation of nonreducing complexes between the analogous swine protein (no sulfhydryls) and cytochrome c suggest that the sulfhydryl group of β-lactoglobulin is the electron donor.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

Acid-Induced Stomatal Opening in Vicia faba L. and the Role of Guard Cell Wall Elasticity

When epidermal peels of Vicia faba L. were treated with solutions of varying pH, stomatal aperture was significantly increased at pH 4.0, 3.0, and 2.7 in darkness, but not in light. This effect was independent of the presence of KCl in the medium. Using a short-term plasmolytic method, estimates were made of the osmotic pressure (II_i) and the volumetric elastic modulus of guard cells, the aperture of which varied due to pretreatments at different pH, in darkness or light. In darkness, the lower pH pretreatments induced an increase in II_i and a decrease in volumetric elastic modulus. In comparison to the response in unbuffered solutions, 10 and 25 millimolar Mes buffer at pH 6.5 significantly reduced the degree of stomatal opening induced by light or by fusicoccin. These results indicate that acid-induced stomatal opening is, at least partially, due to an increase in guard cell wall elasticity which occurs in association with changes in II_i. It is suggested that the observed inhibitory effect of Mes buffer on stomatal opening may be due to a reduction in the degree of acidification of the guard cell wall and a consequent decrease of cell wall elasticity.     

Partitioning of taxifolin-iron ions complexes in octanol-water system

The composition of taxifolin-iron ions complexes in an octanol-water biphasic system was studied using the method of absorption spectrophotometry. It was found that at pH 5.0 in an aqueous biphasic system the complex of [Tf · Fe₂(OH) _k (H₂O)_{8 ? k} ] is present, but at pH 7.0 and 9.0 the complexes of [Tf₂ · Fe(OH) _k (H₂O)_{2 ? k} ] and [Tf · Fe(OH) _k (H₂O)_{4 ? k} ] are predominantly observed. The formation of a stable [Tf₃ · Fe] complex occurred in octanol phase. The charged iron ion of this complex is surrounded by taxifolin molecules, which shield the iron ion from lipophilic solvent. During transition from water to octanol phase the changes of the composition of complexes are accompanied by reciprocal changes in portion of taxifolin and iron ions in these phases. It was shown that the portion of taxifolin in aqueous solution in the presence of iron ions is increased at high pH values, and the portion of iron ions is minimal at pH 7.0. In addition, the parameters of solubility limits of taxifoliniron ions complexes in an aqueous solution were determined. The data obtained gain a better understanding of the role of complexation of polyphenol with metal of variable valency in passive transport of flavonoids and metal ions across lipid membranes.     

Effects of trypsin upon EPR signals arising from components of the donor side of Photosystem II

EPR signals from components functioning on the electron donor side of Photosystem II (PS II) have been monitored in PS II membranes isolated from spinach chloroplasts after treatment with trypsin at pH 7.5 and pH 6.0. The following information has been obtained. (1) The multiline manganese signal, the g = 4.1 signal and Signal II_slow are lost with trypsin treatment at pH 7.5, but not at pH 6.0. (2) At pH 7.5 the multiline S₂ signal and the g = 4.1 signal are lost with approximately the same dependency on the incubation time with trypsin. At pH 6.0 trypsin treatment is known to block electron transfer between Q_A and Q_B (the first and the second quinone electron acceptors, respectively) allowing only a single turnover to occur. Under these conditions both the g = 4.1 signal and the multiline signal are induced by illumination at 200 K and their amplitudes are almost the same as in untreated samples. These results are interpreted as indicating that the g = 4.1 signal arises from a side path donor or from S₂ itself rather than a carrier functioning between the S states and the reaction center as previously suggested. (3) Cytochrome b-559 is converted to its oxidized low-potential form by trypsin treatment at both values of pH. At pH 6.0 the S-state turnover still occurs indicating that the presence of reduced high-potential cytochrome b-559 is not necessary for this process.     

Model complexes for the high spin iron(II) state in the catalytic cycle of cytochrome P450

Studies using ⁵⁷Fe Mössbauer spectroscopy on frozen concentrated solutions of two iron(II) porphyrins in the presence of a large excess of thiophenol or 2-mercaptoethanol are reported. The iron(II) porphyrins used were photoporphyrin IX iron(II) (PPIXFe(II)) and tetra(p-sulphophenyl)porphinato-iron(II) (TTPPSFe(II)). Evidence for high-spin five-coordinate iron(II) complexes were found for both iron(II) porphyrins with thiophenol, but no reaction was found to occur with 2-mecaptoethanol. In contrast to these findings in solutions dilute in TPPSFe(II) evidence was found (from electronic absorption spectroscopy and spectrophotometric titrations) for both thiophenol and 2-mercaptoethanol acting as axial ligands in high-spin five-coordinate TPPSFe(II) species. Mössbauer data for frozen solutions containing TPPSFe(II), carbon monoxide (CO) and either thiol, are consistent with the presence of only low-spin six-coordinate iron(II) complexes. These latter complexes are deduced to contain both a thiol and a CO molecule as axial ligands> These results are discussed in relation to earlier work on PPIXFe(II)-thiol solutions and also in relation to the high-spin iron(II) state in the catalytic cycle of cytochrome P450.The reactions of TPPSFe(II) with excess of either 2-mercaptoethanol or ethyl 2-mercaptoacetate in air are also reported and their complex nature discussed.     

Proton magnetic relaxation and anion effect in solutions of acid ferricytochrome c.

Measurements of the longitudinal relaxation rates of water protons in aqueous solutions of ferricytochrome c and their temperature dependence, were used for the elucidation of the heme iron ligands at acid pH. The relaxation rates increased with a decrease in pH and pK values of 2.5 and 4.48 were evaluated for the aqueous and 6 m urea solutions, respectively. The results at acid pH are compatible with a structure in which two water molecules exchange rapidly between the coordination sphere of high spin heme iron and the bulk. They suggest that concomitantly with the low-high spin transition the histidine-18 and methionine-80 iron bonds break simultaneously. Addition of various anions, including methanesulfonate at pH 1.95 caused a 85% decrease in the net longitudinal relaxation rate. However, neither the chemical shift nor the width of the methyl proton nmr line of methanesulfonate in solution of acid ferricytochrome c were affected indicating that the effect of anions is not due to a direct binding to the heme iron. The relaxation mechanism of the water molecules in the first coordination sphere of the ferric ion in acid cytochrome c is discussed. It appears that the longitudial relaxation rate is modulated by the electronic correlation time of the ferric ion which was calculated to be τ_s = 6 × 10^?11 sec at 60 MHz.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Reactive iron barriers: a niche enabling microbial dehalorespiration of 1,2-dichloroethane

A reactive iron barrier in a contaminated aquifer with low pH was found to dechlorinate 1,2-dichloroethane (1,2-DCA) in situ. This chlorinated ethane is known to resist abiotic reduction by zero valent iron. Samples taken up-gradient and within the barrier were used to inoculate anaerobic batch cultures amended with various electron donors. Cultures inoculated with groundwater from within the reactive iron barrier reduced 1,2-DCA to ethene. The same effect could be achieved by simultaneously supplying hydrogen while neutralising pH. The presence of iron or hydrogen at neutral pH had negligible effects on 1,2-DCA reduction in cultures inoculated with groundwater sampled up-gradient of the barrier. Molecular microbial community characterisation revealed that Dehalobacter species were more abundant in groundwater sampled from within the barrier. These findings suggest reactive iron barriers represent a remediation technology for 1,2-DCA degradation acting through in situ recruitment of 1,2-DCA reducing bacteria such as Dehalobacter.     

The effect of pH on the rate of relaxation of isolated barnacle myofibrillar bundles

CO₂-induced acidosis in barnacle muscle fibres prolongs the relaxation phase of the electrically stimulated contraction (Ashley, C.C., Franciolini, F., Lea, T.J. and Lignon, J. (1979) J. Physiol. 296, 71P). In order to test if this effect is due to a direct action of H⁺ on the relaxation kinetics of the myofilaments, isolated myofibrillar bundles were contracted and relaxed in Ca²⁺ buffer solutions at pH 6.0 and 7.1, in the presence of 20 mM caffeine to inactivate the sarcoplasmic reticulum. At pH 7.1, the relaxation half-time was reduced from 1.5 to 0.3 s as the EGTA concentration in the relaxing solution was progressively increased from 0.3 to 50 mM. The resulting curve was shifted in the direction of increasing EGTA concentration by lowering the pH to 6.0. This effect could be explained by the reduction in affinity of Ca²⁺ for EGTA at pH 6.0, since relaxation half-times for a given relaxing pCa (calculated from the contaminating Ca²⁺ concentrations in the relaxing solutions) were shorter (by about 40%) at pH 6.0 compared with 7.1. However, similar experiments using the new Ca²⁺-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), which is much less pH sensitive than EGTA, indicated that there was no significant difference between relaxation half-times at pH 6.0 and 7.1 for a given relaxing pCa. It is concluded that because no prolongation of relaxation of the myofibrils was observed on lowering the pH from 7.1 to 6.0, the effect of CO₂ on the relaxation of intact muscle fibres is probably due to a modification of sarcoplasmic reticulum activity.     

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.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

α_s1- and β-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (α_s1- and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α_s1- and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.     

The stability of iron(III) complexes formed below pH=3 with glycinate,iminodiacetate, β-hydroxyethyliminodiacetate,N,N-Di-(hydroxyethyl)-glycinate,nitrilotriacetate and triethanolamine

The formation of iron(III) complexes with six monoaminopolycarboxylate and monoaminopoly-alcohol ligands has been investigated by redox and pH measurements in acidic solutions at I=1 (NaClO₄) and 25 °C. The stability of the complexes increases with the number of carboxylate groups and a further enhancement is observed when alcoholic groups are present.     

The iron-quinone acceptor complex in Rhodospirillum rubrum chromatophores studied by EPR

A new EPR signal is reported in Rhodospirillum rubrum chromatophores. The signal is attributed to Q⁻_BFe²⁺, the semiquinone-iron complex of the secondary quinone electron acceptor, on the basis of the following observations. (1) It is induced by a single laser flash given a room temperature and is stable. (2) It is present after odd-numbered flashes and absent after even-numbered flashes when a series of flashes is given. (3) When it is already present, low-temperature illumination results in the disappearance of the signal due to formation of the Q⁻_AFe²⁺Q⁻_B state. (4) Its formation is inhibited by the presence of orthophenanthroline at normal values of pH. The Q⁻_BFe²⁺ signal has two main features, one at g = 1.93 and the other at g = 1.82. The two features have different microwave power and temperature dependences, with the g = 1.82 signal being more difficult to saturate and requiring lower temperatures to be observable. Raising the pH leads to an increase in the g = 1.82 feature, while the g = 1.93 signal decreases in amplitude. It is suggested that the two parts of the signal may represent two EPR forms due to structural heterogeneity. The low-field feature of the Q⁻_BFe²⁺ signal shifts to lower field as the pH is raised and a pK for this change seems to occur at pH 9.4. The Q⁻_AFe²⁺ signal at g = 1.88 also shifts as the pH is increased; however, the shift is less marked than that seen for Q⁻_BFe²⁺, the shift is to higher field and the range over which it occurs is wider and depends upon the temperature of Q⁻_AFe²⁺ formation. This effect may be due to a pK on a protein group being shifted to higher pH by the presence of Q⁻_A. ortho-Phenanthroline broadens and shifts the Q⁻_AFe²⁺ signal. The inhibition of electron transfer between Q⁻_A and Q_B by ortho-phenanthroline becomes less effective at high pH. The new Q⁻_BFe²⁺ signal is unlike other semiquinone-iron signals reported in the literature in bacteria; however, it is remarkably similar to the Q⁻_BFe²⁺ signal reported in Photosystem II.