Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model.

Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the active site structure of isopenicillin N synthase (IPNS) from Cephalosporium acremonium with Cu(II) as a spectroscopic probe. Fourier transform of the stimulated electron spin-echo envelope for the Cu(II)-substituted enzyme, Cu(II)IPNS, revealed two nearly magnetically equivalent, equatorially coordinated His imidazoles. The superhyperfine coupling constant, Aiso, for the remote 14N of each imidazole was 1.65 MHz. The binding of substrate to the enzyme altered the magnetic coupling so that Aiso is 1.30 MHz for one nitrogen and 2.16 MHz for the other. From a comparison of the ESEEM of Cu(II)IPNS in D2O and H2O, it is suggested that water is a ligand of Cu(II) and this is displaced upon the addition of substrate.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule. is the water molecule really necessary for the enzymatic mechanism?

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

Some dicopper complexes of benzimidazole-containing ligands.

Dicopper complexes of the following benzimidazole-containing ligands have been studied as possible models for the active site of hemocyanin: EDTB (N,N,N',N'-tetrakis-(2-benzimidazolylmethyl)-1,2-ethanediamine), EGTB (1,1,10,10-tetrakis-(2-benzimidazolylmethyl)-1,10-diaza-4,7- dioxadecane), and MEGTB (1,1,10,10-tetrakis-(1-methylbenzimidazol-2-y lmethyl)-1,10-diaza-4,7-dioxadecane). The initial oxygenation product of Cu2(EDTB)(ClO4)2 in Me2SO gives optical absorption maxima at 315 nm (epsilon = 3750 M-1 cm-1) and 690 nm (epsilon = 100 M-1 cm-1). The fluorescence emission intensities of Cu2(EDTB)(ClO4)2 at 400 and 700 nm (excitation at 350 nm) decreases rapidly on exposure to air. This suggests oxidation of Cu2(I) to Cu2(II). The x-ray absorption edge spectra suggest that both coppers in the oxygenation product, analyzed as Cu2(EDTB)(ClO4)2(O).3H2O, are Cu(II). From spectrophotometric titration of Cu2(MEGTB)Cl4 with azide, formation constant of the Cu2(MEGTB)N3Cl3 complex has been obtained. Data from cyclic voltammetry experiments suggest that in the presence of azide, Cu(II)(N3)Cu(II) species is present.     

Bacterial cytochrome c nitrite reductase: new structural and functional aspects

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the nitrite reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c nitrite reductase exhibited a remarkably high sulfite reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with nitrite. This potential reaction intermediate of the reduction of nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.     

Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.     

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme

The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.     

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.     

Magnetic resonance studies on the copper site of dopamine beta-monooxygenase in the presence of cyanide and azide anions

In order to elucidate the coordination state of water molecules in the Cu(II) site of dopamine [( 3,4-dihydroxyphenyl)ethylamine] beta-monooxygenase, measurements of the paramagnetic 1H nuclear magnetic relaxation rate of solvent water in the enzyme solution containing cyanide or azide as an exogenous ligand were carried out to obtain the values of intrinsic paramagnetic relaxation rate decrements Rp1 and Rp2 for the ligand-enzyme 1:1 and 2:1 complexes, respectively. Rp1 (percent) values were 53 (pH 5.5) and 52 (pH 7.0) for cyanide and 38 (pH 5.5) and 32 (pH 7.0) for azide, while Rp2 (percent) values were 98 (pH 5.5) and 96 (pH 7.0) for azide. Although no Rp2 values for cyanide were obtained because of its reducing power at the Cu(II) site, the Rp1 and Rp2 values obtained above prove that the Cu(II) center has two coordinated water molecules that are exchangeable for exogenous ligands at either pH. Supporting evidence was provided by electron paramagnetic resonance (EPR) titration, in which the enzyme solution containing cyanide-enzyme (1:1) complex in an equal proportion to uncomplexed enzyme gave an observed paramagnetic relaxation rate decrement, Rp, of 23%. Another characteristic of the Rp1 and Rp2 values was their invariability with respect to pH, indicating that the three-dimensional structure of the Cu(II) site is pH-invariant within the range examined. Binding constants of ligand to enzyme Kb1 and Kb2 for 1:1 and 2:1 complex formation, respectively, were also determined through an analysis of the Rp values; it was found that Kb1 was larger than Kb2 irrespective of pH. (ABSTRACT TRUNCATED AT 250 WORDS)     

The oxidation of 3-hydroxyanthranilic acid by Cu,Zn superoxide dismutase: mechanism and possible consequences

Cu,Zn SOD, but not Mn SOD, catalyzes the oxidation of 3-hydroxyanthranilic acid (3-HA) under aerobic conditions. In the absence of O2, the Cu(II) of the enzyme is reduced by 3-HA. One plausible mechanism involves the reduction of the active site Cu(II) to Cu(I), which is then reoxidized by the O2- generated by autoxidation of the anthranilyl or other radicals on the pathway to cinnabarinate. We may call this the superoxide reductase, or SOR, mechanism. Another possibility invokes direct reoxidation of the active site Cu(I) by the anthranilyl, or other organic radicals, or by the peroxyl radicals generated by addition of O2 to these organic radicals. Such oxidations catalyzed by Cu,Zn SOD could account for the deleterious effects of the mutant Cu,Zn SODs associated with familial amyotrophic lateral sclerosis and of the overproduction or overadministration of wild-type Cu,Zn SOD.     

Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.     

Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling)

The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites are also reflected in the data on binding of the substrate camphor (S) in the presence of the product 5-exo-OH camphor (P) to CYP101 (E). The data are in accordance with the two-site model involving the ternary complex ESP. The affinity of the substrate to the product-bound enzyme as well as the affinity of the product to the substrate-bound enzyme decreased with increase in product concentration, which is consistent with mixed inhibition indicative of inhibitory allosteric interactions between the two sub-sites. Implications of these observations for coupling/uncoupling mechanisms are discussed in the light of the published findings consistent with the two-site behavior of the P450 active site. In addition, kinetic data indicating that the transient high spin intermediate may have to be taken into account for understanding how some P450s have been able to express appreciable hydroxylation activities in the absence of substrate-induced low to high spin transition, observable by the traditional static spectroscopy, are presented.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with guanosine and its derivatives.

Interaction of copper(II) with guanosine, 2'-deoxyguanosine, 1-methylguanosine, 7-methylguanosine and GMP was studied withe use of spectroscopic and magneto-chemical methods. The main site of copper(II) binding in guanosine is nitrogen N-7; participation of N-1 is not excluded. The involvement of carbonyl oxygen in copper binding or copper chelation to N-7 and 0-6 is rather unlikely. A crystalline complex of copper(II) with GMP [Cu(C10H12O8N5P) .(H2O)3] was obtained, and it was demonstrated that copper(II) is bound with N-7 and the phosphate group.     

Evidence that dioxygen and substrate activation are tightly coupled in dopamine beta-monooxygenase. Implications for the reactive oxygen species

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine beta-monooxygenase (DbetaM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DbetaM. First, we have synthesized the substrate analog, beta,beta-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DbetaM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O2* has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-alpha-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DbetaM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DbetaM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DbetaM and peptidylglycine-alpha-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.     

Electron paramagnetic resonance measurements of the ferrous mononuclear site of phthalate dioxygenase substituted with alternate divalent metal ions: direct evidence for ligation of two histidines in the copper(II)-reconstituted protein.

The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites. A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofactor, and a mononuclear iron site is the putative site of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin domain, transfers electrons from NADH to the Rieske center. Any of the metal ions, Fe(II), Cu(II), Co(II), Mn(II), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effecting hydroxylation. Nevertheless, studies of how these metal ions affect both the EPR spectra of the reduced Rieske site and the kinetics of electron transfer in the PDO system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples in which iron of the mononuclear site was replaced with Cu(II). The use of (63)Cu(II), in combination with PDO obtained from cultures grown on media enriched in (15)N [using ((15)NH(4))(2)SO(4) as a sole nitrogen source], [delta,epsilon-(15)N]histidine, as well as natural abundance sources of nitrogen, enabled detailed spectral analysis of the superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines are coordinated to the mononuclear site. Coupled with previous studies [Bertini, I., Luchinat, C., Mincione, G., Parigi, G., Gassner G. T., and Ballou, D. P. (1996) J. Bioinorg. Chem. 1, 468-475] that show the presence of one or two water molecules coordinated to the iron, it is suggested that the mononuclear site is similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, for which crystal structures are available. The lack of observable EPR interaction signals between Cu(II) in the mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 A apart, which is similar to that found in the naphthalene dioxygenase crystal structure.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

Copper(II) complexation by pituitary adenylate cyclase activating polypeptide fragments.

Results are reported from potentiometric and spectroscopic (UV-Vis, CD, and ESR) studies of the protonation constants and Cu2+ complex stability constants of pituitary adenylate cyclase activating polypeptide fragments (HSDGI-NH2, TDSYS-NH2, RKQMAVKKYLAAVL-NH2). With HSDGI-NH2, the formation of a dimeric complex Cu2H-2L2 was found in the pH range 5-8, in which the coordination of copper(II) is glycylglycine-like, while the fourth coordination site is occupied by the imidazole N3 nitrogen atom, forming a bridge between two copper(II) ions. The formation of dimeric species does not prevent the deprotonation and coordination of the amide nitrogen, and in pH above 8 the CuH-2L complex is formed. Aspartic acid in the third position of peptide sequence stabilizes the CuH-2L species and prevents the coordination of a fourth nitrogen donor. Aspartic acid residue in the second position of TDSYS-NH2 stabilizes the CuL (2N) complex but does not prevent deprotonation and binding of the second and third peptide nitrogens to give 3N and 4N complexes at higher pH. The tetradecapeptide amide forms with copper(II) ions unusually stable 3N and 4N complexes compared to pentaalanine amide.