Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy

Nitric oxide synthase (EC 1.14.13.39; NOS) converts L-arginine into NO and L-citrulline in a two-step reaction with Nomega-hydroxy-L-arginine (NOHLA) as an intermediate. The active site iron in NOS has thiolate axial heme-iron ligation as found in the related monooxygenase cytochrome P450. In NOS, tetrahydrobiopterin (BH4) is an essential cofactor for both steps, but its function is controversial. Previous optical studies of the reaction between reduced NOS with O2 at -30 degrees C suggested that BH4 may serve as an one-electron donor in the first cycle, implying formation of a trihydrobiopterin radical. We investigated the same reaction under identical conditions with electron paramagnetic resonance spectroscopy. With BH4-containing full-length neuronal NOS we obtained an organic free radical (g-value 2.0042) in the presence of Arg, and a similar radical was observed with the endothelial NOS oxygenase domain in the presence of Arg and BH4. Without substrate the radical yield was greatly (10x) diminished. Without BH4, or with NOHLA instead of Arg, no radical was observed. With 6-methyltetrahydropterin or 5-methyl-BH4 instead of BH4, radicals with somewhat different spectra were formed. On the basis of simulations we assign the signals to trihydropterin radical cations protonated at N5. This is the first study that demonstrates the formation of a protonated trihydrobiopterin radical with the constitutive isoforms of NOS, and the first time the radical was obtained without exogenous BH4. These results offer strong support for redox cycling of BH4 in the first reaction cycle of NOS catalysis (BH4 <--> BH3.H+).     

Hunting oxygen complexes of nitric oxide synthase at low temperature and high pressure

The reaction of nitric oxide synthase (NOS) with oxygen is fast and takes place within several steps, separated by ephemeral intermediates. The use of extreme experimental conditions, such as low temperature and high pressure, associated to rapid kinetic analysis, has proven to be a convenient tool to study this complex reaction. Stopped-flow experiments under high pressure indicated that oxygen binding occurred in more than one step. This was further corroborated by the detection of two short-lived oxy-compounds, differing in their spectral and electronic properties. Oxy-I resembles the ferrous oxygen complex known for cytochrome P450, whereas oxy-II appears to be locked in the superoxide form. Subzero temperature spectroscopy, together with an analytical separation method, revealed that the subsequent one-electron reduction of the oxygen complex is carried out by the NOS cofactor tetrahydrobiopterin (BH4). The low-temperature stabilized oxidation product of BH4 was found to be a protonated BH3 radical. Finally, work in the presence of a BH4 analog indicated that proton transfer to the activated oxygen complex is a second essential function of BH4.     

High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin.

The crystal structures of the catalytic domain (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH) in its catalytically competent Fe(II) form and binary complex with the reduced pterin cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) have been determined to 1.7 and 1.5 A, respectively. When compared with the structures reported for various catalytically inactive Fe(III) forms, several important differences have been observed, notably at the active site. Thus, the non-liganded hPheOH-Fe(II) structure revealed well defined electron density for only one of the three water molecules reported to be coordinated to the iron in the high-spin Fe(III) form, as well as poor electron density for parts of the coordinating side-chain of Glu330. The reduced cofactor (BH4), which adopts the expected half-semi chair conformation, is bound in the second coordination sphere of the catalytic iron with a C4a-iron distance of 5.9 A. BH4 binds at the same site as L-erythro-7,8-dihydrobiopterin (BH2) in the binary hPheOH-Fe(III)-BH2 complex forming an aromatic pi-stacking interaction with Phe254 and a network of hydrogen bonds. However, compared to that structure the pterin ring is displaced about 0.5 A and rotated about 10 degrees, and the torsion angle between the hydroxyl groups of the cofactor in the dihydroxypropyl side-chain has changed by approximately 120 degrees enabling O2' to make a strong hydrogen bond (2.4 A) with the side-chain oxygen of Ser251. Carbon atoms in the dihydroxypropyl side-chain make several hydrophobic contacts with the protein. The iron is six-coordinated in the binary complex, but the overall coordination geometry is slightly different from that of the Fe(III) form. Most important was the finding that the binding of BH4 causes the Glu330 ligand to change its coordination to the iron when comparing with non-liganded hPheOH-Fe(III) and the binary hPheOH-Fe(III)-BH2 complex.     

Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin

Endothelial nitric-oxide synthase (eNOS) plays important roles in vascular physiology and homeostasis. Whether eNOS catalyzes nitric oxide biosynthesis or the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite is dictated by the bioavailability of tetrahydrobiopterin (BH(4)) and L-arginine during eNOS catalysis. The effect of BH(4) and L-arginine on oxygen-induced radical intermediates has been investigated by single turnover rapid-freeze quench and EPR spectroscopy using the isolated eNOS oxygenase domain (eNOS(ox)). Three distinct radical intermediates corresponding to >50% of the heme were observed during the reaction between ferrous eNOS(ox) and oxygen. BH(4)-free eNOS(ox) produced the superoxide radical very efficiently in the absence of L-arginine. L-Arginine decreased the formation rate of superoxide by an order of magnitude but not its final level or EPR line shape. For BH(4)-containing eNOS(ox), only a stoichiometric amount of BH(4) radical was produced in the presence of L-arginine, but in its absence a new radical was obtained. This new radical could be either a peroxyl radical of BH(4) or an amino acid radical was in the vicinity of the heme. Formation of this new radical is very rapid, >150 s(-1), and it was subsequently converted to a BH(4) radical. The trapping of the superoxide radical by cytochrome c in the reaction of BH(4)(-) eNOS(ox) exhibited a limiting rate of approximately 15 s(-1), the time for the superoxide radical to leave the heme pocket and reach the protein surface; this reveals a general problem of the regular spin-trapping method in determining radical formation kinetics. Cytochrome c failed to trap the new radical species. Together with other EPR characteristics, our data strongly support the conclusion that this new radical is not a superoxide radical or a mixture of superoxide and biopterin radicals. Our study points out distinct roles of BH(4) and L-arginine in regulating eNOS radical intermediates. BH(4) prevented superoxide formation by chemical conversions of the Fe(II)O(2) intermediate, and l-arginine delayed superoxide formation by electronic interaction with the heme-bound oxygen.     

Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.     

Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine,and its implications for the mechanism of catalysis and substrate activation

Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of L-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH-Fe(II).BH(4).THA) of the catalytically active Fe(II) form of a truncated form (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and 3-(2-thienyl)-L-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131-155, where the maximum r.m.s. displacement (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH-Fe(II).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for L-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving Tyr138 from a surface position into the active site, may reflect a possible functional role for this residue.     

Two modes of binding of N-hydroxyguanidines to NO synthases: first evidence for the formation of iron-N-hydroxyguanidine complexes and key role of tetrahydrobiopterin in determining the binding mode

The interaction of various N-alkyl- and N-aryl-N'-hydroxyguanidines with recombinant NOS containing or not containing tetrahydrobiopterin (BH(4)) was studied by visible, electronic paramagnetic resonance (EPR), and resonance Raman (RR) spectroscopy. N-Hydroxyguanidines interact with the oxygenase domain of BH(4)-free inducible NOS (BH(4)-free iNOS(oxy)), depending on the nature of their substituent, with formation of two types of complexes that are characterized by peaks around 395 (type I) and 438 nm (type II') during difference visible spectroscopy. The complex formed between BH(4)-free iNOS(oxy) and N-benzyl-N'-hydroxyguanidine 1 (type II') exhibited a Soret peak at 430 nm, EPR signals at g = 1.93, 2.24, and 2.38, and RR bands at 1374 and 1502 cm(-)(1) that are characteristic of a low-spin hexacoordinated Fe(III) complex. Analysis of its EPR spectrum according to Taylor's equations indicates that the cysteinate ligand of native BH(4)-free iNOS(oxy) is retained in that complex. Similar iron(III)-ligand complexes were formed upon reaction of 1 and several other N-hydroxyguanidines with BH(4)-free full-length iNOS and BH(4)-free nNOS(oxy). However, none of the tested N-hydroxyguanidines were able to form such iron(III)-ligand complexes with BH(4)-containing iNOS(oxy), indicating that a major factor involved in the mode of binding of N-hydroxyguanidines to NOS is the presence (or absence) of BH(4) in their active site. Another factor that plays a key role in the mode of binding of N-hydroxyguanidines to NOS is the nature of their substituent. The N-hydroxyguanidines bearing an N-alkyl substituent exclusively or mainly led to type II' iron-ligand complexes. Those bearing an N-aryl substituent mainly led to type II' complexes, even though some of them exclusively led to type I complexes. Interestingly, the K(s) values calculated for BH(4)-free iNOS(oxy)-N-hydroxyguanidine complexes were always lower when their substituents bore an aryl group (140-420 microM instead of 1000-3900 microM), suggesting the existence of pi-pi interactions between this group and an aromatic residue of the protein. Comparison of the spectral and physicochemical properties of the N-hydroxyguanidine complexes of BH(4)-free iNOS(oxy) (type II') with those of the previously described corresponding complexes of microperoxidase (MP-8) suggests that, in both cases, N-hydroxyguanidines bind to iron(III) via their oxygen atom after deprotonation or weakening of the O-H bond. The aforementioned results are discussed in relation with recent data about the transient formation of iron-product intermediates during the catalytic cycle of l-arginine oxidation by eNOS. They suggest that N-hydroxyguanidines could constitute a new class of good ligands of heme proteins.     

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

1-Methyl-4-phenylpyridinium-induced apoptosis in cerebellar granule neurons is mediated by transferrin receptor iron-dependent depletion of tetrahydrobiopterin and neuronal nitric-oxide synthase-derived superoxide

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP(+) toxicity. Results show that MPP(+) treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP(+) caused a time-dependent depletion of tetrahydrobiopterin (BH(4)) that was mediated by H(2)O(2) and transferrin iron. Depletion of BH(4) decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP(+)-mediated "uncoupling" of nNOS decreased *NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH(4) biosynthesis or cell-permeable iron chelator and TfR antibody to prevent iron-catalyzed BH(4) decomposition inhibited MPP(+) cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP(+)-induced iron uptake, BH(4) depletion, proteasomal inactivation, and apoptosis. We conclude that MPP(+)-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH(4), leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed.     

Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.     

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.     

Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), l-citrulline, NADP+, and water from l-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and l-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or l-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and l-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or l-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and l-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without l-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new approximately 75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only l-arginine in nNOSox (without BH4 but with l-Arg) caused conversion of approximately 70% of superoxide radical to a novel radical, explaining how l-arginine decreases the level of superoxide production in nNOSox (without BH4 but with l-Arg). The regulatory role of l-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gr?slund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.     

Deferiprone protects against doxorubicin-induced myocyte cytotoxicity

The iron chelating hydroxypyridinone deferiprone (CP20, L1) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were examined for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. The results of this study showed that both deferiprone and dexrazoxane were able to protect myocytes from doxorubicin-induced lactate dehydrogenase release. Deferiprone quickly and efficiently removed iron(III) from its complex with doxorubicin. In addition, this study also showed that deferiprone rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex, suggesting that in the myocyte, deferiprone should also be able to displace iron from its complex with doxorubicin. It was shown by electron paramagnetic resonance spectroscopy that under hypoxic conditions myocytes were able to reduce doxorubicin to its semiquinone free radical. Deferiprone also greatly reduced hydroxyl radical production by the iron(III)-doxorubicin complex in the xanthine oxidase/xanthine superoxide generating system. Together these results suggest that deferiprone may protect against doxorubicin-induced damage to myocytes by displacing iron bound to doxorubicin, or chelating free or loosely bound iron, thus preventing site-specific iron-based oxygen radical damage.     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.