Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of native and type-2-copper-depleted Vietnamese-lacquer-tree and Japanese-lacquer-tree laccases.

The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.     

Isolation of mannose-binding proteins from human and rat liver

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.     

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.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.     

Presence of coupled trinuclear copper cluster in mammalian ceruloplasmin is essential for efficient electron transfer to oxygen

The reactivity with dioxygen of a mammalian (sheep) ceruloplasmin, anaerobically reduced with ascorbate, was found to depend on the state of the Type 2 and Type 3 copper centers, as monitored by EPR and optical spectroscopy. A complete reoxidation by air after anaerobic reduction with ascorbate was observed with samples (A) purified by the single-step procedure described for chicken ceruloplasmin (Calabrese, L., Carbonaro, M., and Musci, G. (1988) J. Biol. Chem. 263, 6480-6483), while samples prepared by traditional multistep procedure (B) or subjected to freeze-thawing (C) displayed partial and very slow reoxidation, reflecting the functional nonequivalence of blue coppers which is considered a typical property of mammalian ceruloplasmin. The rate of reduction of the 330 nm chromophore was found to increase as a function of the extent and rate of reoxidation of different samples, while the 610 nm band displayed an opposite trend. Samples B and C showed a Type 2 copper signal in the EPR spectrum, while sample A showed practically no Type 2 copper in the oxidized protein, and a transient Type 2-like signal during reduction. The presence of a trinuclear Type 2-Type 3 cluster can therefore be proposed for all ceruloplasmins, and the integrity of the copper-copper coupling is essential for efficient oxidase behavior.     

Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.     

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.     

Presence of reduced type 1 copper in ceruloplasmin as revealed by reaction with hydrogen peroxide

The reaction of hydrogen peroxide with ox or sheep ceruloplasmin leads to approximately 10% increase of the optical absorption band at 610 nm and of the Type 1 EPR signal. No inactivation or denaturation of the protein is apparent up to 15 H2O2 molar excess. Oxygen is able to restore about 50% of the Type 1 copper absorption in ascorbate-reduced ceruloplasmin, while the other half is recovered after addition of H2O2. It appears that H2O2 undergoes a specific redox reaction with ceruloplasmin, which reveals a fraction of the total copper to be present in the native protein as reduced copper. This fraction is apparently Type 1 copper, while Type 2 is not affected by H2O2.     

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.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

Reassessment of copper stoichiometry in ascorbate oxidase

A very pure ascorbate oxidase solution was obtained by dissolving a crystalline sample of the enzyme. The ratio between 280 and 610 nm absorbancies was 22.5. It contained 8.0 +/- 0.2 Cu ions, 50% EPR detectable, per dimeric molecule (140,000 M.W.) with a molar extinction coefficient of 10,000 cm-1 at 610 nm. Two Cu ions were removed by treatment with N,N-diethyldithiocarbamate. The optical blue absorption band was unaffected, while two EPR detectable Cu ions were lost, with disappearance of the type 2 Cu signal. It is concluded that native ascorbate oxidase contains two type 1, two type 2, and four type 3 Cu ions.     

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.     

Spectral study of ascorbate oxidase

The electronic, CD and EPR spectra of ascorbate oxidase isolated from the green zucchini squash (Cucurbita pepo medullosa) in 0.1 M phosphate buffer (pH 6.8) have been investigated. The visible absorption bands are clearly resolved in the CD spectrum, where the extrema occur at 735, 610, 550, 475 and 330 nm, while weak additional CD activity possibly occurs near 420 nm. The near-UV spectrum is dominated by the absorption of the aromatic amino acid residues centered at 280 nm, while resolved CD bands occur at 296, 291, 283, 265 and 240 nm. In the far-UV region the protein CD spectrum reflects its secondary structure: a single negative maximum at 218 nm suggests a predominant anti-parallel β conformation for ascorbate oxidase. The frozen solution EPR spectrum of the protein has been fitted according to a new computer simulation procedure. The following parameters were obtained: for the type 1 copper g_z = 2.222, g_x = 2.032, g_y = 2.056, A_z = 59 G, A_x = 11 G, and A_y = 5 G; for the type 2 copper g ? = 2.240, g_⊥ = 2.057, A? = 179 G and A_⊥ = 1 G. Of the eight copper atoms present in the protein four are EPR-detectable: three of type 1 and one of type 2, as shown by computer simulation of the EPR spectrum. Ascorbate oxidase is a rather unstable protein when purified and it is sensitive to a number of environmental factors. Aging of the protein leads to a decrease in the ratio between the type 1 and type 2 coppers. A new species formed at the early stages of the aging process, that has been spectrally characterized, suggests that the loss of the type 1 copper is preceded by a change in the symmetry of the original type 1 site from pseudotetrahedral to pseudotetragonal.     

The Type 3 copper site is intact but labile in Type 2-depleted laccase

We report results of experiments designed to characterize the Type 1 and Type 3 copper sites in Rhus laccase depleted of Type 2 copper (T2D). Use of the Lowry method for determining protein concentration yielded the value 5620 +/- 570 M-1 cm-1 for the extinction of the 615-nm absorption band of this protein. Anaerobic reductive titrations with Ru(NH)3)6(2)+ and Cr(II)aq ions established the presence of three electron-accepting centers, which are reduced in a complex manner. Treatment of T2D laccase with a 70-fold excess of H2O2 induced a new shoulder at 330 nm (delta epsilon = 660 M-1 cm-1), as well as intensity perturbations at 280 and 615 nm. Comparison of difference spectra show that this 330-nm band derives from a Type 3 copper-bound peroxide and not from a reoxidized Type 3 site. Dioxygen reoxidation of ascorbate-reduced T2D laccase produced new difference bands at 330 nm (delta epsilon = 770 M-1 cm-1) and 270 nm (delta epsilon = 13,000 M-1 cm-1), the former assigned to a bound peroxide which is a dioxygen reduction intermediate. In the corresponding epr spectrum of this material new Cu(II) g parallel features (A parallel approximately 130 G) indicative of an isolated copper ion and a triplet signal near 3,400 G were observed, originating from the Type 3 sites of separate T2D laccase molecules. Reoxidation by ferricyanide or by dioxygen as mediated by iron hexacyanide did not produce these changes. Thus the magnetism of the reoxidized Type 3 site in T2D laccase can be perturbed as a consequence of aerobic turnover. The suggestion is advanced that there are presently three forms of T2D laccase, possibly metastable conformational isotypes, accounting for the apparently contradictory reports on the properties of this protein.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding

Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].