Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.     

Coordination environment for the type 3 copper center of tree laccase and CuB of cytochrome c oxidase as determined by electron nuclear double resonance

A new rhombic EPR signal was recently discovered in the partially reduced type 2 copper-depleted Rhus vernicifera laccase (Reinhammar, B. (1983) J. Inorg. Biochem., in press). The signal originates from one of the type 3 Cu(II) ions that becomes EPR-detectable as a result of the selective reduction of the other copper ion in the exchange-coupled Cu(II)-Cu(II) pair. The 14N and 1H and 63,65Cu electron nuclear double resonance (ENDOR) of this uncoupled Cu(II) now have been collected and represent the first ENDOR measurements of a type 3 copper site. The data indicate that the copper is coordinated by at least three nitrogenous ligands, at least one of which is an imidazole. H/D exchange suggests a nearby H2O or OH-, perhaps as a fourth ligand. A similar EPR signal is seen for CuB of reduced cytochrome c oxidase under turnover conditions. The 14N ENDOR, and, therefore, the structure, of this site corresponds extremely closely to that of the laccase type 3 (Cu(II).     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.     

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactions of nitric oxide with tree and fungal laccase

The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.     

Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.     

Rapid freeze- and chemical-quench studies of dopamine beta-monooxygenase: comparison of pre-steady-state and steady-state parameters

The copper-containing enzyme dopamine beta-monooxygenase has been studied with regard to pre-steady-state kinetics of tyramine hydroxylation and reduction of enzyme-bound Cu2+ by chemical- and freeze-quench EPR techniques. The bulk of the enzyme-bound copper (approximately 70%) is reduced in a single-exponential process with a limiting rate constant of 250 s-1, Km = 0.9 mM, consistent with participation of both copper ions in the redox events of catalysis. The remaining copper is reduced much more slowly (k approximately 2 s-1) or not at all, attributed to a distribution of copper into inhibitory binding sites and the presence of some inactive enzyme. Knowledge of the Cu2+ reduction rate, together with rate constants calculated from steady-state isotope effects [Miller, S. M., & Klinman, J. P. (1985) Biochemistry 24, 2114-2127], has allowed prediction of pre-steady-state product formation transients. Measurement of these transients under conditions of excess ascorbate shows close agreement with prediction, supporting the validity of individual rate constants obtained from steady-state data. Kinetic modeling shows further that the predominant steady-state enzyme form is the enzyme-product complex (E-P), which is expected to show a correspondingly large (approximately 70% of total copper) EPR signal for bound Cu2+. Surprisingly, the steady state is characterized by a low (19% of total copper) EPR signal. This lack of correlation between the anticipated and observed steady-state EPR signal suggests either antiferromagnetic coupling in binuclear copper centers or reduction of Cu2+ in this enzyme form by ascorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center

Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.     

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.     

超氧化物歧化酶活性中心中铜离子的氧化还原研究

用电子顺磁共振EPR技术研究铜锌超氧化物歧化酶(Cu·Zn-SOD)与底物(O_2~(·-)反应达到平衡态时铜离子的EPR波谱表明,在平衡态时的铜离子处于还原态。用还原剂H_2O_2、NaBH_4处理Cu·Zn-SOD后,酶活力变化不同,电泳行为也不同。用NaBH_4处理SOD其活性及电泳行为接近天然酶,但经H_2O_2还原后的酶活性损失严重,电泳后出现多条色带。     

Azide binding to the trinuclear copper center in laccase and ascorbate oxidase.

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.     

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition.

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out ability to generate this species to study reactions of a3+(3) with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O2 and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a3+(3) reacts within less than 10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a3+(3) species responds to O2 and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a3+(3) registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a3+(3) in either case. In all these experiments, in which EPR detectable a3+(3) was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a3+(3) to other 'activated' species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.     

Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers

Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.     

Characterization of the potentiometric behavior of soluble cytochrome oxidase by magnetic circular dichroism. Evidence in support of heme-heme interaction

Magnetic circular dichroism spectroscopy has been used to characterize the oxidation-reduction behavior of cytochromes a and a3 during potentiometric experiments. The experiments were complicated by the presence of slow, time-dependent changes during reduction, and it appears that reliable results can only be obtained during reoxidation or with enzyme that has been subjected to a cycle of reduction and oxidation. Under all experimental conditions the reduction levels of cytochrome a and a3 are comparable. This result cannot be reconciled with a model in which the two heme centers have defined and well resolved potentials. The most straightforward explanation of the data requires an oxidation-reduction coupling of the potentials of the two hemes, i.e. an allosteric or heme-heme interaction, which is about 2 K cal/mol in magnitude. There is a good correlation between magnetic circular dichroism and EPR measurements obtained on parallel samples. The kinetics of the slow, time-dependent processes have been characterized by measurement of a variety of spectral properties and enzyme activity. All parameters measured change at comparable rates, implying a common rate-controlling event. A new copper EPR signal has been observed at high pH. This signal appears to rise from the "EPR-undetectable" copper center.     

The type 2 copper of ascorbate oxidase

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at −30°C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500–3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at −30°C the activity fell to 1400–2000 units in the phosphate buffer but only to 2600–2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at −30°C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu²⁺ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu²⁺ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)₆³⁻, large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.     

The reduction of fungal laccase at high pH

1. 1. The nature and mechanism of the reduction of fungal laccase (p-diphenol: O₂ oxidoreductase, EC 1.10.3.2) obtained on an increase in pH have been studied by optical and electron paramagnetic resonance (EPR) spectroscopy and by measurements of O₂ concentration.

2. 2. The decreases in the optical absorption and the EPR signal of the “blue” Type 1 Cu²⁺ at high pH indicate that this ion is reduced. This is confirmed by oxidation with hexachloroiridate(IV) which restores the blue color. The “nonblue” Type 2 Cu²⁺ remains divalent over the pH range studied, as seen from the EPR spectra.

3. 3. Approximately one equivalent of hexachloroiridate(IV) is sufficient to restore the color of a pH-bleached protein which suggests that the reduction involves a single electron. A comparison between the optical spectra at pH 5 and 8 shows that the two-electron accepting unit, which at pH 5.5 is reduced concomitantly with the Type 1 Cu²⁺, remains oxidized in the protein brought to high pH. This unit can be reduced at pH 8.3 by octacyanotungstate(IV), as shown by the fact that this reductant in anaerobic titrations is found to add about two electrons (and no more) to a protein already having the Type 1 copper reduced. Thus, an increase in pH introduces a difference in the reduction behavior of the electron acceptors in fungal laccase.

4. 4. Oxygraph experiments show that there is no production of O₂ with an increase in pH, as would occur if water was oxidized by laccase. On the contrary, there is a continuous consumption of O₂ at both pH 5 and 8, indicating that the protein preparation contains a reducing substance which is responsible for the pH-dependent reduction.

The reductive half-reaction of xanthine oxidase. Identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine.

The reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (also called 2-oxo-6-methylpurine) has been studied under both anaerobic and aerobic conditions. Reaction of enzyme with substoichiometric concentrations of hydroxymethylpurine in aerobic 0.1 M 3-(cyclohexylamino)propanesulfonic acid, 0.1 N KCl, 0.3 mM EDTA, pH 10.0, exhibits two reaction intermediates detectable by UV-visible spectrophotometry. The rate constants for formation of the first intermediate, conversion of the first to the second, and the decay of the second to give oxidized enzyme are 18, 1.2, and 0.13 s-1, respectively. The difference spectra of these two intermediates relative to oxidized enzyme are characterized by absorbance maxima at 470 and 540 nm, respectively, with extinction changes (relative to oxidized enzyme) of approximately 410 M-1 cm-1. The 0.13 s-1 decay of the second intermediate agrees well with kcat of 0.11 s-1 determined under the same conditions. Based on a comparison of the kinetics of the reaction as monitored by UV-visible absorption and electron paramagnetic resonance spectrometry, it is concluded that these spectral intermediates arise from the molybdenum center of the enzyme in the MoIV and MoV valence states, respectively, the latter corresponding to the species exhibiting the "very rapid" MoV EPR signal known to be formed in the course of the reaction. This conclusion is supported by the results of experiments using cytochrome c reduction to follow the formation of superoxide production in the course of the aerobic reaction of xanthine oxidase with substoichiometric hydroxymethylpurine, which demonstrate unequivocally that the species exhibiting the very rapid EPR signal is formed by one-electron oxidation of a MoIV species rather than direct one-electron reduction of MoVI by substrate. No evidence is found for the formation of any of the MoV EPR signals designated "rapid" in the present studies, and it is concluded that this species is not a bona fide catalytic intermediate in the reductive half-reaction of xanthine oxidase.     

EPR studies of spin-labeled bovine plasma amine oxidase: the nature of the substrate-binding site.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.     

Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135.

The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO(2) (-). Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme.     

Trapping a hydrazine reduction intermediate on the nitrogenase active site

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.