Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.     

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5-50 Fe(2+)/HoSF established that the reaction is overall first order in Fe(2+) concentration. However, when full progress curves were analyzed at a variety of Fe(2+)/HoSF ratios, two first-order reactions (k(1) approximately 0.035 s(-1) and k(2) approximately 0.007 s(-1)) were found to contribute to the overall Fe(2+) oxidation reaction. The proportion of the fast reaction increased with increasing Fe(2+)/HoSF ratio until at approximately 400, it was the dominant reaction. For the Fe(2+)/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe(2+) oxidation. Comparison of successive additions of 1.0 Fe(2+)/HoSF showed that oxidation of the first 8-10 Fe(2+) produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H(+)/Fe(2+) ratio confirmed this difference in behavior by giving an H(+)/Fe(2+) ratio of approximately 1.0 below and 2.0 for ratios >30 Fe(2+)/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.     

Important role of tetrahydrobiopterin in no complex formation and interdomain electron transfer in neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is composed of a heme oxygenase domain and a flavin-bound reductase domain. Ca(2+)/calmodulin (CaM) is essential for interdomain electron transfer during catalysis, whereas the role of the catalytically important cofactor, tetrahydrobiopterin (H4B) remains elusive. The product NO appears to bind to the heme and works as a feedback inhibitor. The present study shows that the Fe(3+)-NO complex is reduced to the Fe(2+)-NO complex by NADPH in the presence of both l-Arg and H4B even in the absence of Ca(2+)/CaM. The complex could not be fully reduced in the absence of H4B under any circumstances. However, dihydrobiopterin and N(G)-hydroxy-l-Arg could be substituted for H4B and l-Arg, respectively. No direct correlation could be found between redox potentials of the nNOS heme and the observed reduction of the Fe(3+)-NO complex. Thus, our data indicate the importance of the pterin binding to the active site structure during the reduction of the NO-heme complex by NADPH during catalytic turnover.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi.

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.     

Kinetics of iron oxidation by Leptospirillum ferriphilum dominated culture at pH below one

The kinetics of ferrous iron oxidation by Leptospirillum ferriphilum (L. ferriphilum) dominated culture was studied in the concentration range of 0.1-20 g Fe(2+)/L and the effect of ferric iron (0-60 g Fe(3+)/L) on Fe(2+) oxidation was investigated at pH below one. Denaturing gradient gel electrophoresis of PCR amplified 16S rRNA genes followed by partial sequencing confirmed that the bacterial community was dominated by L. ferriphilum. In batch assays, Fe(2+) oxidation started without lag phase and the oxidation was completed within 1 to 60 h depending on the initial Fe(2+) concentration. The specific Fe(2+) oxidation rates increased up to around 4 g/L and started to decrease at above 4 g/L. This implies substrate inhibition of Fe(2+) oxidation at higher concentrations. Haldane equation fitted the experimental data reasonably well (R(2) = 0.90). The maximum specific oxidation rate (q(m)) was 2.4 mg/mg VS . h, and the values of the half saturation (K(s)) and self inhibition constants (K(i)) were 413 and 8,650 mg/L, respectively. Fe(2+) oxidation was competitively inhibited by Fe(3+) and the competitive inhibition constant (K(ii)) was 830 mg/L. The time required to reach threshold Fe(2+) concentration was around 1 day and 2.3 days with initial Fe(3+) concentration of 5 and 60 g/L, respectively. The threshold Fe(2+) concentration, below which no further Fe(2+) oxidation occurred, linearly increased with increasing initial Fe(2+) and Fe(3+) concentrations. Fe(2+) oxidation proceeds by L. ferriphilum dominated culture at pH below 1 even in the presence of 60 g Fe(3+)/L. This indicates potential of using and biologically regenerating concentrated Fe(3+) sulfate solutions required, for example, in indirect tank leaching of ore concentrates.     

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.     

Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.

Superoxide dismutase (SOD) from extracts of anaerobically maintained Bacteroides thetaiotaomicron was a dimer of equally sized 23,000-molecular-weight monomers joined noncovalently. A preparation with a specific activity of 1,200 U/mg contained 1.1 g-atom of Fe, 0.6 g-atom of Zn, and less than 0.05 g-atom of Mn per mol of dimer. The apoprotein, prepared by dialysis of iron-SOD in 5 M guanidinium chloride-20 mM 8-hydroxyquinoline, had no superoxide-scavenging activity when renatured without exogenous metal. Enzymatic activity was restored to the denatured apoprotein by dialysis against either 1 mM Fe(NH4)2 or 1 mM MnCl2 in 20 mM Tris (pH 7.0). The Fe-reconstituted enzyme and the native enzyme were inhibited approximately 50% by 0.2 mM NaN3, whereas the Mn-reconstituted enzyme was inhibited 60% by 10 mM NaN3. Aeration of the anaerobic cells resulted in a fourfold induction of an azide-resistant SOD. The enzyme (43,000 molecular weight) isolated from aerated cells was a dimer of equally sized subunits. The metal content was 1.0 g-atom of Mn, 0.55 g-atom of Fe, and 0.3 g-atom of Zn per mol of dimer. Enzymatic activity of the denatured apoprotein from this enzyme was also restored on addition of either iron or manganese. The constitutive Fe-SOD and the O2-induced Mn-SOD, tested alone and in combination, migrated identically on acrylamide gels, had similar amino acid compositions, and had alanine as the sole N-terminal amino acid. These data are consistent with the synthesis of a single apoprotein in either anaerobically maintained or oxygenated cells. We have observed a similar phenomenon with SOD from Bacteroides fragilis (E. M. Gregory, Arch. Biochem. Biophys. 238:83-89, 1985).     

Ca2+/calmodulin-dependent activation and inactivation mechanisms of alphaCaMKII and phospho-Thr286-alphaCaMKII

Thr(286) autophosphorylation is important for the role of alphaCaMKII in learning and memory. Phospho-Thr(286)-alphaCaMKII has been described to have two types of activity: Ca(2+)-independent partial activity and Ca(2+)/calmodulin-activated full activity. We investigated the mechanism of switching between the two activities in order to relate them to the physiological functioning of alphaCaMKII. Using a fluorometric coupled enzyme assay and smooth muscle myosin light chain (MLC) as substrate, we found that (1) Ca(2+)-independent activity of phospho-Thr(286)-alphaCaMKII represents 5.0 (+/-3.7)% of the activity measured in the presence of optimal concentrations of Ca(2+) and calmodulin and (2) Ca(2+) in the presence of calmodulin activates the enzyme with a K(m) of 137 (+/-56) nM and a Hill coefficient n = 1.8 (+/-0.3). In contrast, unphosphorylated alphaCaMKII has a K(m) for Ca(2+) in the presence of calmodulin of 425 (+/-119) nM and a Hill coefficient n = 5.4 (+/-0.4). Thus, the activity of phospho-Thr(286)-alphaCaMKII is essentially Ca(2+)/calmodulin dependent with MLC as substrate. In physiological terms, our data suggest that alphaCaMKII is only activated in stimulated neurones whereas Ca(2+)/calmodulin activation of phospho-Thr(286)-alphaCaMKII can occur in resting cells (approximately 100 nM [Ca(2+)]). Stopped-flow experiments using Ca(2+)/TA-cal [Ca(2+)/2-chloro-(epsilon-amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin] showed that at 100 nM [Ca(2+)] partially Ca(2+)-saturated Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complexes existed. These are likely to account for the activity of the phospho-Thr(286)-alphaCaMKII enzyme at resting [Ca(2+)]. Ca(2+) dissociation measurements by a fluorescent Ca(2+) chelator revealed that the limiting Ca(2+) dissociation rate constants were 1.5 s(-1) from the Ca(2+)/cal.alphaCaMKII and 0.023 s(-1) from the Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complex, accounting for the differences in the Ca(2+) sensitivities of the Ca(2+)/cal.alphaCaMKII and Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII enzymes.     

Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system.     

Uncoupled ATP hydrolysis and thermogenic activity of the sarcoplasmic reticulum Ca2+-ATPase: coupling effects of dimethyl sulfoxide and low temperature

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.     

An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A

The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A ( J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.     

Class-1 hemoglobin and antioxidant metabolism in alfalfa roots

In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked to antioxidant metabolism and affects the cellular redox level. The influence of Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H₂O₂ and ascorbate was investigated in alfalfa root cultures transformed to over-express (Hb+) or down-regulate (Hb–) class-1 Hb. Hb+ lines had substantially increased ascorbate levels as well as elevated monodehydroascorbate reductase and ascorbate peroxidase activities. Hb– lines showed significant increases in dehydroascorbate reductase and glutathione reductase activities. The observed changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both at high (40 kPa) and low (3 kPa) O₂ pressures. Hb– lines had significantly reduced levels of the NO- and H₂O₂-sensitive enzyme, aconitase, as compared to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb– lines, as treatment of plant extracts with the NO-donor DEANO also affected aconitase activity. The H₂O₂ levels were not significantly different amongst the lines and showed no variation with change in oxygen partial pressure. In conclusion, the expression of class-1 Hb improves the antioxidant status through increased ascorbate levels and increased activity of enzymes involved in H₂O₂ removal.     

Comparison of the energetics of lactose active transport: artificial versus enzyme-associated energy source

To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated. The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate). The oxidation of an electron donor provided the energy necessary for the transport process. The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane. Part of the oxidation energy was utilized to form delta mu H+. Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm). Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+. These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source. Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized. The oxidation reaction in the presence of membrane vesicles was discussed.     

The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.     

Ferrous ion binding to recombinant human H-chain ferritin. An isothermal titration calorimetry study

Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe(2+) binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe(2+) binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe(2+) travels to the dinuclear ferroxidase center prior to its oxidation to Fe(3+). Calorimetric titrations show that the ferroxidase center is the principal locus for Fe(2+) binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe(2+). That only one site of the ferroxidase center is occupied by Fe(2+) implies that Fe(2+) oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 microM), only 12 Fe(2+)/protein bind at pH 6.51 increasing to 24 Fe(2+)/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe(2+) to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe(2+) binding. The total Fe(2+) binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D131I+E134F), indicating that the primary avenue by which Fe(2+) gains access to the interior of ferritin is through these eight channels. The binding stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe(2+) binding are similar for the two with an average values (DeltaH degrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M(-)(1) at pH 7.04). Since channel mutations do not completely prevent Fe(2+) binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.     

Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase

A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.