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1.
Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence. 相似文献
2.
H2O2 was shown to reduce the copper ion of native bovine Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) (ECu2+) and to oxidize the reduced enzyme (ECu+). The time-course of these processes was monitored by NMR measurement of the longitudinal relaxation rate of the water protons. A steady-state characterized by the same ratio [ECu2+]/[( EC2+] + [ECu+]) was obtained either by starting from the oxidized or the reduced enzyme. The kinetics of these processes appear to be quite complex, since different reactions between H2O2, or its reaction products, and the enzyme-bound copper control the reaction rate. The solution of the differential equations describing the kinetic processes showed that the oxidation and the reduction of the copper ion by H2O2 are first-order with respect to the copper ion itself only when these processes approach the steady-state. The rate constants of the reduction and oxidation reactions were calculated according to these equations and were found to have comparable values which are in the range 5-80 and 5-45 M-1.min-1, respectively, changing the pH from 5.6 to 7 at 0.21 M ionic strength. This result, together with the dependence of the reaction rates on pH and ionic strength, points to HO2- as the reactive species in both processes, and indicates that the electrostatic control of the access of the peroxide to the active site is the rate-determining step of the two redox reactions. 相似文献
3.
Fet3p is a multicopper oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O(2) to H(2)O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O(2) reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O(2), the type 1 Cu Cys(484) was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His(126)Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His(126)Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O(2) to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond. 相似文献
4.
An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin. 总被引:1,自引:1,他引:0
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The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids. 相似文献
5.
Bryan J. Johnson Erik T. Yukl Valerie J. Klema Judith P. Klinman Carrie M. Wilmot 《The Journal of biological chemistry》2013,288(39):28409-28417
The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I). 相似文献
6.
Dell'acqua S Pauleta SR Monzani E Pereira AS Casella L Moura JJ Moura I 《Biochemistry》2008,47(41):10852-10862
The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center. 相似文献
7.
Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper. 总被引:4,自引:0,他引:4
1. The type 2 copper in Rhus vernicifera laccase was completely removed without loss of other types of copper. The properties of this protein derivative and the role of type 2 copper in the catalytic action of laccase was investigated. 2. The molar extinction coefficient at 614 nm of the blue chromophore decreases from 5700 to 4700 cm-1 on removal of type 2 copper. There are no apparent absorption changes at other wavelengths in the visible or near ultraviolet region when this copper is taken away. The electron-paramagnetic-resonance (epr) parameter A parallel and the linewidth of type 1 Cu2+ decreases on removal of type 2 copper. 3. The rate of reduction of type 1 Cu2+ is not affected by removal of type 2 copper but the reduction of the two-electron acceptor is greatly impaired. These results strongly support the idea that type 1 Cu2+ is the primary site for electron transfer between substrate and enzyme and that the two-electron acceptor in the native enzyme is reduced by simultaneous electron transfer from reduced types 1 and 2 copper. 4. Reoxidation of types 1 and 3 copper and the formation of the oxygen intermediate are the same processes in native and type-2-depleted enzyme. These observations suggests that type 2 copper is not involved in the formation and rapid decay of the oxygen intermediate and that it is not necessary for the stabilization of this intermediate. 5. Two new epr signals are observed on reoxidation of reduced type-2-depleted laccase. One is temporarily formed on re-reduction of reoxidized enzyme and it is suggested that it might arise from copper, possibly type 3 copper. The other one is stable for hours and it is proposed that it might come from a modified oxygen intermediate. 相似文献
8.
The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/Km[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins. 相似文献
9.
Akiyama N Nakanishi I Ohkubo K Satoh K Tsuchiya K Nishikawa T Fukuzumi S Ikota N Ozawa T Tsujimoto M Natori S 《Journal of biochemistry》2007,142(1):41-48
N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37 degrees C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H(2)O(2) is produced via the formation of superoxide anion O2(.-) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H(2)O(2) production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H(2)O(2). 相似文献
10.
A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase 总被引:4,自引:0,他引:4
Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested. 相似文献
11.
Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas. 相似文献
12.
T Ookawara N Kawamura Y Kitagawa N Taniguchi 《The Journal of biological chemistry》1992,267(26):18505-18510
Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction. 相似文献
13.
Ellis MJ Prudêncio M Dodd FE Strange RW Sawers G Eady RR Hasnain SS 《Journal of molecular biology》2002,316(1):51-64
Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme. 相似文献
14.
High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants, D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to better than 2.0 A resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type II copper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weak electron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N delta 2 atom of Asn98 to form a hydrogen bond with the bound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIR structure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soaked H255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite binding site despite the lack of direct interaction with the substrate in the native enzyme. The resulting pentacoordinate copper site in the H255N structure also serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated to the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soaked D98N and H255N crystal structures may represent an evolutionary link between the mononuclear type I copper centers and dinuclear Cu(A) sites. 相似文献
15.
The copper amine oxidases (CAOs) catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain in the active site. Monooxygenation within the ring of tyrosine at a single Cu(II) site is unique in biology and occurs as an early step in the formation of TPQ. The mechanism of this reaction has been further examined in the CAO from Hansenula polymorpha (HPAO). When a Clark electrode fitted to a custom-made, gastight apparatus over a range of initial concentrations of O(2) was used, rates of O(2) consumption at levels greater than air are seen to be reduced relative to earlier results, yielding K(D)(apparent) = 216 microM for O(2). This is consistent with a mechanism in which O(2) binds reversibly to the active site, triggering a conformational change that promotes ligation of tyrosinate to Cu(II). The activated Cu(II)-tyrosinate species has been proposed to react with O(2) in a rate-limiting step, although it was also possible that breakdown of a putative peroxy-intermediate controlled TPQ formation. To test the latter hypothesis, Cu(II)-free HPAO was prepared with 3,5-ring-[(2)H(2)]-tyrosine incorporated throughout the primary sequence. The absence of an isotope effect on the rate of TPQ formation eliminates cleavage of this C-H bond in a proposed Cu(II)-aryl-peroxide intermediate as a rate limiting step. The role of methionine 634, previously found to moderate O(2) binding during the catalytic cycle, is shown here to serve a similar function in TPQ formation. As with catalysis, the rate of TPQ formation correlates with the volume of the hydrophobic side chain at position 634, implicating similar binding sites for O(2) during catalysis and cofactor biogenesis. 相似文献
16.
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. 相似文献
17.
Agaricus bisporus metapotyrosinase: preparation, characterization, and conversion to mixed-metal derivatives of the binuclear site 总被引:1,自引:0,他引:1
The alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase undergo inactivation in the presence of oxalate. The inactivation rate law is first order in enzyme and second order in oxalate. On a more rapid time scale than inactivation, oxalate acts as a competitive inhibitor of the catecholase reaction of tyrosinase. After removal of oxalate by dialysis, the inactivated enzyme is found to contain 50% of the original copper, all of which is present as paramagnetic, mononuclear copper sites. The ESR parameters of this copper indicate a tetragonal environment with nitrogen and oxygen ligands. The product of oxalate inactivation has lost one copper from each binuclear site and is thus a metapo derivative. Addition of Cu(II) to metapotyrosinase results in complete recovery of copper and catalytic activity. Prolonged storage of metapotyrosinase, in the absence of any additional Cu(II), results in copper migration, producing a 50% recovery of the original specific activity, expressed on a protein basis. Copper migration converts metapo sites into equal numbers of reconstituted, holo sites and fully apo sites. Both copper migration and copper reconstitution follow apparent first-order kinetics and are pH dependent. The involvement of two ionizable groups accounts for the observed pH dependence of each process. For copper migration pKa values of 6.0 and 8.8 were found, while for copper reconstitution the pKa values were 5.4 and 6.9. Addition of either Co(II) or Zn(II) to metapotyrosinase results in the formation of enzymatically inactive, mixed-metal derivatives of the binuclear copper site having one Cu(II) and one Co(II) or Zn(II) ion.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
We report here the first detailed study of the dithionite reduction kinetics of a copper-containing dissimilatory nitrite reductase (NiR). The reduction of the blue type 1 copper (T1Cu) center of NiR preparations that contained both type 1 and type 2 copper atoms, followed biphasic kinetics. In contrast, NiR that was deficient in type 2 copper (T2DNiR), followed monophasic kinetics with a second-order rate constant (T2D)k = 3.06 x 10(6) m(-1) s(-1). In all cases the SO(2)(.-) radical rather than S(2)O(4)(2-) was the effective reductant. The observed kinetics were compatible with a reaction mechanism in which the T1Cu of the fully loaded protein is reduced both directly by dithionite and indirectly by the type 2 Cu (T2Cu) site via intramolecular electron transfer. Reduction kinetics of the T2Cu were consistent with SO(2)(.-) binding first to the T2Cu center and then transferring electrons (112 s(-1)) to reduce it. As SO(2)(.-) is a homologue of NO(2)(-), the NiR substrate, it is not unlikely that it binds to the catalytic T2Cu site. Effects on the catalytic activity of the enzyme using dithionite as a reducing agent are discussed. Reduction of the semireduced T1Cu(I)T2Cu(II) state followed either second-order kinetics with k(2) = 3.33 x 10(7) m(-1) s(-1) or first-order kinetics with 52.6 s(-1) < (T1red)k(1) < 112 s(-1). Values of formation constants of the T1Cu(II)T2Cu(II)-SO(2)(.-) and T1Cu(I)T2Cu(II)-SO(2)(.-) adducts showed that the redox state of T1Cu affected binding of SO(2)(.-) at the catalytic T2Cu center. Analysis of the kinetics required the development of a mathematical protocol that could be applied to a system with two intercommunicating sites but only one of which can be monitored. This novel protocol, reported for the first time, is of general application. 相似文献
19.
Svistunenko DA 《Biochimica et biophysica acta》2001,1546(2):365-378
The reaction of hydrogen peroxide H(2)O(2) with horse heart metmyoglobin (HH metMb), sperm whale metmyoglobin (SW metMb) and human metHb (metHbA) was studied at pH 6-8 by low temperature (10 K) EPR spectroscopy with the emphasis on the peroxyl radicals formed during the reaction. The same type of peroxyl radical was found in both myoglobin systems, as was concluded from close similarities in the spectroscopic properties of the radicals and in their kinetic dependences. This is consistent with previous reports of the peroxyl radical being localised on the Trp14 of SW and HH myoglobins. There are two types of peroxyl radical found in the metHbA/H(2)O(2) system, one (ROO-I) having spectral parameters, kinetic and pH dependences similar to those of the peroxyl radical found in both myoglobin systems. The other peroxyl radical (ROO-II) found in metHbA treated with H(2)O(2) has slightly different, though distinguishable, spectral parameters and a significantly different kinetic dependence as compared to those of the peroxyl radical common for all three proteins studied (ROO-I). The concentration of ROO-I radical formed in the three proteins on addition of H(2)O(2) correlates with the effectiveness of incorporating molecular oxygen into styrene oxide reported before for these three proteins. It is shown that a different distance from Trp14 to haem iron in the three proteins might be the structural basis for the different yield of the peroxyl radical and the different efficiency of incorporation of molecular oxygen into styrene. The site of the peroxyl radical found only in metHbA (ROO-II) is speculated to be the Trp37 residue of the beta-subunit of HbA. 相似文献
20.
The x-ray absorption for copper and zinc in oxidized and reduced superoxide dismutase, as well as in various model compounds, was studied. Upon reduction of the protein, the added electron affects the copper site almost exclusively, while the zinc remains virtually unchanged. Reduction decreases the charge on the copper atom [toward Cu(I)] and changes the configuration of the copper site so that it becomes less symmetric. An analysis of the copper absorption observed with the oxidized enzyme and a comparison with that for Cu(II)(imid)4 suggests that the copper is not simply ligated to four imidazoles. The addition of H2O2 to superoxide dismutase reduces the copper to Cu(I), while oxygen addition to the peroxide-reduced protein restores the copper to Cu(II). 相似文献