RESUMEN
The binding of Mn2+ to manganese-depleted photosystem II was investigated after chemical modification of histidyl and carboxylic acid residues in the presence or absence of the native manganese cluster. K(M) values for Mn2+ were determined from steady-state electron transfer between Mn2+ and 2,6-dichlorophenolindophenol, the dissociation constant for Mn2+ was measured by observing the effect of added Mn2+ on the reduction of the primary donor P680+ after a saturating flash, and single-turnover electron donation from Mn2+ was followed by monitoring the decay kinetics of the EPR signal from the flash-induced tyrosine Zox radical. K(M) values for Mn2+ were found to be highly pH-dependent in both modified and unmodified photosystem II membranes. Treatment with histidine modifiers after removal of the manganese complex increased the K(M) values between 2.5 and 10 times and increased the dissociation constant for Mn2+ 8-fold, compared to membranes that were modified in the presence of the manganese cluster. Modification of carboxylic acid residues after removal of the manganese cluster increased the K(M) about 5-fold compared to membranes that were modified in the presence of the manganese cluster. The reduction rate of tyrosine Zox by Mn2+ was diminished after modification of either histidine or carboxylic acid residues. The apparent second-order rate constant decreased from 2.6 x 10(6) M(-1) s(-1) to 0.05 x 10(6) M(-1) s(-1) after histidine modification in the presence or absence of manganese, to 0.77 x 10(6) M(-1) s(-1) after carboxylic acid residue modification in the presence of manganese, and to 0.18 x 10(6) M(-1) s(-1) after carboxylic acid modification in the absence of manganese. Our results indicate the existence of two different manganese binding sites containing histidine, and at least two manganese sites with carboxylic acid residues, which are differently shielded against modifying agents by the native manganese cluster.
Asunto(s)
Histidina , Manganeso/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , 2,6-Dicloroindofenol/farmacología , Benzoquinonas/farmacología , Sitios de Unión , Ácidos Carboxílicos , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Concentración de Iones de Hidrógeno , Cinética , Luz , Complejo de Proteína del Fotosistema II , Spinacia oleraceaRESUMEN
Photosystem II membranes, dialyzed against a Cl(-)-free buffer to remove bound Cl-, lost about 65% of the control activity. A light-intensity study of the Cl(-)-free membranes showed that all PS II centers were able to evolve oxygen at about 35% of the control rate when measured in Cl(-)-free medium. The Cl(-)-depleted membranes were immediately (< 15 s) reactivated to 85-90% of the original activity by the addition of fairly high concentrations of Cl- (Kd = 0.5 mM), but both Cl- and the activity were promptly lost when the membranes immediately after reactivation were diluted in a Cl(-)-free medium. However, stabilization of Cl(-)-binding could be accomplished by prolonged incubation in the presence of Cl-. The transition to stable binding, followed using 36Cl-, occurred over several minutes. The stable binding was further characterized by a Kd of 20 microM and a t1/2 for dissociation of about 1h [Lindberg et al. (1993) Photosynth. Res. 38, 401-408]. The effects on S2 signals of removal of Cl- were studied using EPR. The depletion of Cl- was accompanied by a shift in intensity toward the g = 4.1 signal at the expense of the multiline signal. When Cl- or Br- but not F- was added to the depleted PS II membranes, the original distribution of the signals was immediately (< 30 s) restored. We propose that Cl(-)-binding responsible for high oxygen-evolution activity and normal EPR properties of the S2 state may occur either as high affinity (Kd = 20 microM) and slowly exchanging (t1/2 = 1 h), or as low affinity (Kd = 0.5 mM) and rapidly exchanging (t1/2 < 15 s). Our results suggest that Br- but not F- has a mode of binding similar to that of Cl-. The high-affinity state is the normal state of binding, but once Cl- has been removed, it will first rebind as low-affinity, rapidly exchanging followed by conversion into a high-affinity, slowly exchanging mode of binding.
Asunto(s)
Aniones/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Unión Competitiva , Bromuros/química , Bromuros/metabolismo , Sistema Libre de Células , Cloruros/química , Cloruros/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Fluoruros/química , Fluoruros/metabolismo , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II , Unión ProteicaRESUMEN
The functional properties and the content of non heme iron and cytochrome b559 were investigated by measuring flash induced transient changes of the relative fluorescence quantum yield and applying Mössbauer spectroscopy. It was found that untreated PS II membrane fragments contain a heterogeneous population of two types of non heme iron centers and about 2 cytochrome b559 per PS II. Twofold treatment of these samples with a recently described 'iron depletion' procedure (MacMillan, F., Lendzian, F., Renger, G. and Lubitz, W. (1995) Biochemistry 34, 3144-3156) leads to a complete loss (below the detection limit of Mössbauer spectroscopy) of the non heme iron center while more than 50% of the PS II complexes retain the functional integrity for light induced formation of the 'stable' radical pair Y(OX)(Z) P680Pheo Q(-.)(A). This sample type deprived of virtually all non heme iron in PS II provides a most suitable material for magnetic resonance studies that require an elimination of the interaction between Fe2+ and nearby radicals.
Asunto(s)
Grupo Citocromo b/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II , Spinacia oleracea/metabolismo , Grupo Citocromo b/química , Grupo Citocromo b/aislamiento & purificación , Ferrocianuros/farmacología , Membranas Intracelulares/metabolismo , Hierro/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Teoría Cuántica , Espectrometría de Fluorescencia , Espectroscopía de Mossbauer/métodosRESUMEN
The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Sitios de Unión , Radioisótopos de Calcio , Cinética , Manganeso/metabolismo , Peso Molecular , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II , Conformación Proteica , Spinacia oleracea/metabolismoRESUMEN
Reaction centers from Rhodobacter sphaeroides R-26 were treated with trypsin in the dark and during illumination (in the charge-separated state). Trypsination resulted in a time-dependent modification of the reaction centers, reflected in changes in the charge recombination rate, in the inhibition of QA- to QB electron transfer, and eventually to inhibition of charge separation. Comparisons of centers with ubiquinone or anthraquinone in the QA site, in which the charge recombination pathways are different, indicate that trypsination affects charges close to the QA(-)-binding site. Studies of light-induced voltage changes from moving charges in reaction centers incorporated in lipid layers on a Teflon film, a technique which allows the discrimination of effects on donor and acceptor sides, indicate that the acceptor side is preferentially degraded by trypsin in the dark. Tryptic digestion during illumination generally resulted in a marked strengthening and acceleration of the effects seen already during dark treatment, but new effects were also detected in gel electrophoretic peptide patterns, in optical spectra, and in the kinetic measurements. Optical kinetic measurements revealed that the donor side of the reaction centers became susceptible to modification by trypsin during illumination as seen in the value of the binding constant for soluble cytochrome c2 which increased by a factor of 2, whereas it was much less affected after trypsination of reaction centers in the dark. The influence of illumination on the rate and mode by which trypsin acts on reaction centers indicates that changes in the protein conformation follow charge separation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Tripsina , Grupo Citocromo c/aislamiento & purificación , Grupo Citocromo c/metabolismo , Citocromos c2 , Oscuridad , Electroquímica , Transporte de Electrón , Electroforesis en Gel de Poliacrilamida , Cinética , Luz , Modelos Teóricos , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Espectrofotometría , Termodinámica , Factores de TiempoAsunto(s)
Calcio/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Spinacia oleracea/metabolismo , Sitios de Unión , Tampones (Química) , Radioisótopos de Calcio , Citratos , Ácido Cítrico , Ácido Egtácico , Cloruro de Magnesio , Complejo de Proteína del Fotosistema II , Cloruro de SodioRESUMEN
Photosystem II (PS II) particles isolated from spinach in the presence of 10 µM CuSO4 contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO4 was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1-1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.
RESUMEN
(36)Cl(-) was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of (36)Cl(-), saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10(-4) s(-1) at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on (36)Cl(-), which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br(-) and NO3 (-). The slowly exchanging Cl(-) did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.
RESUMEN
A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Transporte de Electrón , Concentración de Iones de Hidrógeno , Cinética , Liposomas/metabolismo , Concentración Osmolar , Oxidación-Reducción , Oxígeno/metabolismoRESUMEN
EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal IIf and decreased the EPR signal from state S2. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal IIf, but did not relieve the decrease in the state S2 signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal IIf, Z+ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.
Asunto(s)
Organoides/metabolismo , Fotosíntesis , Plantas/metabolismo , Oscuridad , Espectroscopía de Resonancia por Spin del Electrón , Oxígeno/metabolismo , Sales (Química) , TermodinámicaRESUMEN
A new purification procedure for the water-soluble 33 kDa protein of Photosystem II is presented. The method is based on the selective release of the 33 kDa protein at slightly elevated temperatures and involves a minimum of purification steps. Starting with spinach leaves, the pure protein may be obtained in about 4 h, with a yield usually higher than 60%.
Asunto(s)
Cloroplastos/análisis , Proteínas de Plantas/aislamiento & purificación , Aminoácidos/análisis , Fenómenos Químicos , Química , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Fotosíntesis , Solubilidad , AguaRESUMEN
The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (Rönnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.
Asunto(s)
Azidas/metabolismo , Cianuros/metabolismo , Citocromo-c Peroxidasa/metabolismo , Fluoruros/metabolismo , Peroxidasas/metabolismo , Pseudomonas aeruginosa/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Conformación ProteicaRESUMEN
The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.
Asunto(s)
Citocromo-c Peroxidasa/metabolismo , Peroxidasas/metabolismo , Pseudomonas aeruginosa/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Congelación , Hemo/metabolismo , Cinética , Oxidación-ReducciónRESUMEN
In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu2+ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from CuB2+, the copper which is EPR-nondetectable in the resting enzyme. Optical absorption changes in the 500-700 nm region accompanies the decay of the new Cu2+ EPR signal. Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.
Asunto(s)
Cobre , Complejo IV de Transporte de Electrones , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-ReducciónRESUMEN
A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed. With Rhus laccase, the new signal is shown to originate from one of the copper ions that are nondetectable in the resting enzyme, and evidence is presented for the signals in Polyporus laccase and cytochrome c oxidase also stemming from the metal pairs that are antiferromagnetically coupled in the oxidized enzymes. The new signals show strong rhombic character, and the EPR parameters place them in a category different from the signals of type 1 as well as of type 2 Cu2+ ions.
Asunto(s)
Basidiomycota/enzimología , Cobre/análisis , Complejo IV de Transporte de Electrones/análisis , Oxidorreductasas/análisis , Plantas Tóxicas , Polyporaceae/enzimología , Toxicodendron/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Lacasa , Especificidad de la EspecieRESUMEN
The reaction of soluble mixed-valence-state (a3+CuA 2+.CuB + A32+) cytochrome oxidase with O2 at low temperature was studied by optical and e.p.r. spectroscopy. The existence of three intermediates [Clore & Chance (1978) Biochem. J. 173, 799-8101] was confirmed. From the e.p.r data it is clear that cytochrome a and CuA remain in the low-spin ferric and cupric states respectively throughout the reaction. No e.p.r. signals attributable to cytochrome a3 or CuB were seen in the intermediates. The difference spectra (intermediates minus unliganded mixed-valence-state cytochrome oxidase) and absolute spectra of the three intermediates were obtained. The chemcal nature of the three intermediates is discussed in terms of their spectroscopic properties. A catalytic cycle for cytochrome oxidase is proposed.
Asunto(s)
Complejo IV de Transporte de Electrones , Oxígeno , Fenómenos Químicos , Química , Frío , Cobre , Espectroscopía de Resonancia por Spin del Electrón , Modelos Químicos , Oxidación-Reducción , EspectrofotometríaRESUMEN
The reaction of fully reduced soluble bovine heart cytochrome oxidase with O2 at 173K was investigated by low-temperature optical and e.p.r. spectroscopy, and the kinetics of the reaction were analysed by non-linear optimization techniques. The only e.p.r. signals seen during the course of the reaction are those attributable to low-spin cytochrome a3+ and CuA2+. Quantitative analysis of e.p.r. signals shows that, at the end point of the reaction at 173K, nearly 100% of CuA is in the cupric state but only about 40% of cytochrome a is in the ferric low-spin state. The optical spectra recorded at this stage of the reaction show incomplete oxidation of haem and the absence of a 655 nm absorption band. The only reaction scheme that accounts for both the e.p.r. and optical data is a four-intermediate mechanism involving a branching pathway. The reaction is initiated when fully reduced cytochrome oxidase reacts with O2 to form intermediate I. This is then converted into either intermediate IIA or intermediate IIB. Of these, intermediate IIB is a stable end product at 173 K, but intermediate IIA is converted into intermediate III, which is the stable state at 173 K in this branch of the mechanism. The kinetic analysis of the e.p.r. data allows the unambiguous assignments of the valence states of cytochrome a and CuA in the intermediates. Intermediate I contains cytochrome a2+ and CuA+, intermediate IIA contains low-spin cytochroma a3+ and CuA+, intermediate IIB contains cytochrome a2+ and CuA2+, and intermediate III contains low-spin cytochrome a3+ and CuA2+. The electronic state of the O2-binding CuBa3 couple during the reoxidation of cytochrome oxidase is discussed in terms of an integrated structure containing CuB, cytochrome a3 and O2.
Asunto(s)
Complejo IV de Transporte de Electrones , Oxígeno , Fenómenos Químicos , Química , Frío , Cobre , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Modelos Químicos , Oxidación-Reducción , EspectrofotometríaRESUMEN
1. The reaction of the electron acceptors in Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) have been studied with stopped-flow and rapid-freeze EPR techniques. The studies have been directed mainly towards elucidation of the role of the type 2Cu2+ as a possible pH-sensitve regulator of electron transfer. 2. Anaerobic reduction experiments with Rhus laccase indicate that the type 1 and 2 sites contribute one electron each to the reduction of the two-electron-accepting type 3 site. There is also evidence that the reduction of the type 1 Cu2+ triggers the reduction of the type 2 Cu2+. 3. Only at pH values at which the reduction of the two-electron acceptor is limited by a slow intramolecular reaction can an OH- be displaced from the type 2 Cu2+ by the inhibitor F-. 4. A model describing the role of the electron-accepting sites in catalysis is formulated.
Asunto(s)
Catecol Oxidasa/metabolismo , Anaerobiosis , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Cinética , Plantas Tóxicas , Toxicodendron/enzimologíaRESUMEN
The reactions of Rhus vernicifera (monophenol,dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) with the reducing substrates hydroquinone and ascorbic acid have been investigated with the stopped-flow technique. Rhus laccase appears to be present in two molecular forms with a pH-sensitive equilibrium constant regulating the relative concentrations of each species. A model for the reaction of Rhus laccase with reducing substrates has been formulated. The model is similar to one formulated earlier for the anaerobic reduction of laccase from Polyporus versicolor (Andréasson, L.-E., Malström, B.G., Strömberg, C. and Vänngård, T. (1973) Eur. J. Biochem. 34, 434-439) and accounts for the reduction also of this enzyme. The essentials of the model are as follows: Electrons are taken up from reductants one at a time. The type 1 Cu2+ has a central role in mediating the transfer of at least one of the electrons needed for the reduction of the co-operative two-electron acceptor. Intramolecular reactions determine the concentrations of two molecular forms of the enzyme and influence the rate of reduction of the two-electron acceptor. The model, which has been used for successful simulations of the anaerobic reduction of Rhus laccase, is capable of explaining the reduction of laccases also in the presence of the inhibitor F-. In addition, the model gives an explanation of the behaviour of the laccases when reducing substrates and O2 are simultaneously present and is consistent with earlier observations of the post-steady-state reduction of the type 1 Cu2+ and the two-electron accetor (Holwerda, R.A. and Gray, H.B. (1974) J. Am. Chem. Soc. 96, 6008-6022).
Asunto(s)
Catecol Oxidasa/metabolismo , Plantas/enzimología , Anaerobiosis , Ácido Ascórbico , Transporte de Electrón , Fluoruros/farmacología , Concentración de Iones de Hidrógeno , Hidroquinonas , Cinética , Oxidación-Reducción , Oxígeno , Plantas Tóxicas , Toxicodendron/enzimologíaRESUMEN
1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.