Heterogeneity of the Type 3 copper in Japanese-lacquer-tree (Rhus vernicifera) laccase.

The two steps of the titration of the Japanese-lacquer-tree (Rhus vernicifera) laccase with N3- [Morpurgo, Rotilio, Finazzi-Agrò & Mondovi (1974) Biochim. Biophys. Acta 336, 324-328; LuBien, Winkler, Thamann, Scott, Co, Hodgson & Solomon (1981) J. Am. Chem. Soc. 103, 7014-7016] were shown to be two distinct reactions, each involving one different portion of the native enzyme molecules. The difference consists in the oxidation state of the Type 3 Cu, which is reduced in the portion with higher affinity for N3- and oxidized in the portion with lower affinity for N3-. The difference is eliminated by treatment with oxidizing (H2O2) or reducing agents, and a single N3- adduct is then formed. The e.p.r. spectra of the H2O2-treated enzyme and of its F- derivatives support this interpretation of the results. The similarity of the spectroscopic properties of the high-affinity N3- adduct to those of the N3- adducts of half-met-haemocyanins and half-met-tyrosinase is discussed.     

Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper.

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.     

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.     

The removal of the type-2 copper from Rhus vernicifera laccase.

We have studied the removal of the type-2 copper from tree laccase (Rhus vernicifera) by treatment with EDTA at pH 5.2 in the presence of a redox buffer containing ferri- and ferrocyanide. The efficiency with which the copper is removed depends on the Fe(CN) 6(4-)/Fe(CN) 6(3-) ratio. We have varied this ratio from approx. 2:1 to about 50:1 and the best results were obtained with the highest ratio, i.e., the most cathodic solution potential. Nevertheless, the presence of Fe(CN) 6(3-) is required for the procedure to be effective. Although we cannot exclude the possibility that a mixed-valence form of laccase is the reactive species, we believe the results are better explained by a model which assumes that the removal of the type-2 copper depends upon an ordered sequence of oxidation-reduction reactions. Specifically, we propose that the copper is released as the monovalent ion from previously reduced laccase and then reoxidized in solution and sequestered with EDTA. The reoxidation step drives the reaction because recombination with the protein is inhibited when copper is in the divalent form. In testing this model, we have also shown that the type-2 copper can be removed under strictly reducing conditions when 4,4'-dicarboxy-2,2'-biquinoline (BCA) is present to complex the copper(I) ion. Although the BCA method is effective, the reaction takes longer, perhaps because of the limited solubility of BCA at the pH values of interest. Finally, we have found that the best results are obtained with either method when a cyanometalate ion such as Fe(CN) 6(3-) or Co(CN) 6(3-) is present in the medium. The exact role of this factor has yet to be established, but there is no indication that free cyanide has a role in the process. The most likely interpretation is that some type of binding interaction with the protein facilitates copper release.     

Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism.

The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).     

Peroxide binding to the type 3 site in Rhus vernicifera laccase depleted of type 2 copper

The interaction between hydrogen peroxide and oxidized $\text{Rhus}\text{vernicifera}$ laccase from which the type 2 copper has been removed, was investigated. For that end, the circular dichroic spectrum of the modified enzyme has been measured in the presence of increasing concentrations of hydrogen peroxide. The characteristic band observed upon binding peroxide to native laccase is also observed for the type 2 copper depleted enzyme. However, there are several quantitative differences in the latter one. First, the intensity is lower and band width is larger. Secondly, from the titrations, it becomes apparent that the affinity for H₂O₂ is markedly lower than that of the native enzyme. While the affinity for the native enzyme is higher than 10⁸ M^?1, it decreases to 1·10⁴ M^?1 for the type 2 depleted enzyme.     

Temperature and anation studies of the type 2 site in Rhus vernicifera laccase

Temperature-dependent structural changes involving the type 2 site in laccase are probed by EPR studies of a derivative of laccase in which the type 1 Cu has been replaced by Hg(II) [Morie-Bebel, M. M., Morris, M. C., Menzie, J. L., & McMillin, D. R. (1984) J. Am. Chem. Soc. 106, 3677-3678]. At the temperature extremes (123 and 299 K), single well-defined species are present, but at intermediate temperatures (between 213 and 253 K), the presence of multiple structures is indicated. For the first time, the room temperature EPR spectrum of the type 2 copper has been resolved. Azide binding and fluoride binding have also been studied as a function of temperature. The results suggest that each anion preferentially interacts with the type 3 site in fluid solution and that these adducts can be trapped by rapidly cooling the sample to 123 K. Annealing the adducts at 253 K permits rearrangement and binding at an equatorial position of the type 2 Cu. This pathway to anation at the type 2 site contrasts sharply with previous studies which required a large excess of anions, and it reveals important insight into the flexibility of the type 2/type 3 cluster in laccase.     

Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore.

1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.     

1H NMR of native and azide-inhibited laccase from Rhus vernicifera

The 1H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time. These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in the high frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differences between the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structural properties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over the T1 copper ligands. Overall, these data demonstrate that 1H NMR can be fruitfully applied to characterize the electronic properties of the metal sites of blue oxidases at room temperature.     

Peroxide and redox titrations of type 2 copper depleted laccase

The chemistry of Type 2 copper depleted T2D Rhus laccase has been investigated with regard to the binding of peroxide, and the ability of the enzyme to undergo reduction and reoxidation. Although the peroxide affinity is diminished in the T2D enzyme (10⁴ M^?1) relative to the holo-enzyme ((? 10⁸ M^?1) the actual mode of binding as a Type 3 μ-peroxo complex remains, as indicated by absorption and CD spectral measurements. Anaerobic reductive and reoxidative titrations with hydroquinone and hydrogen peroxide respectively revealed that the Type 3 copper pairwise interaction is disrupted during reduction but can be restored on reoxidation. The concept of separate Type 2 and Type 3 copper redox centers is suggested to be inadequate in view of the loss of functional integrity by the Type 3 site on removal of Type 2 copper.     

Oxidative titrations of Rhus vernicifera laccase and its specific interaction with hydrogen peroxide.

The reaction of oxidized $\text{Rhus}\text{vernicifera}$ laccase and H₂O₂ leads specifically to the formation of a stable, high affinity complex. It is characterized by an absorption band at 325 nm and is most probably formed with the type 3 site. Oxidative titrations of laccase show a different pathway from the reductive ones. This is also expressed in different Nernst coefficients observed for each half of the redox cycle (2 for reduction, 1 for oxidation). Oxidation of the type 3 site by H₂O₂ proceeds in a bimolecular reaction, whereas type 1 is oxidized in an indirect pathway.     

Kinetic studies of Rhus vernicifera laccase. Role of the metal centers in electron transfer.

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., Malstr?m, B.G., Str?mberg, C. and V?nng?rd, 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).     

Effects of laser radiation on rhus vernicifera laccase,type 2 Cu-depleted laccase,and stellacyanin

Laser irradiation in the 450 nm region brings about irreversible changes in the copper sites of Rhus vernicifera laccase and its type 2 Cu-depleted derivative. The absorption band at 614 nm disappears after ~ 2 hr of irradiation with a 200 mW laser beam; the amount of the paramagnetic detectable copper does not decrease, indicating no reduction of these types of copper. No apparent rearrangement of the protein backbone occurs, as ultaviolet dichroic spectra of the enzyme before and after the irradiation do not show appreciable differences. Stellacyanin is insensitive to laser radiation at any wavelength.     

Spectroscopic and kinetic studies on the oxygen-centered radical formed during the four-electron reduction process of dioxygen by Rhus vernicifera laccase.

The oxygen-centered radical bound to the trinuclear copper center was detected as an intermediate during the reoxidation process of the reduced Rhus vernicifera laccase with dioxygen and characterized by using absorption, stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies and by super conducting quantum interface devices measurement. The intermediate bands appeared at 370 nm (epsilon approximately 1000), 420 nm (sh), and 670 nm (weak) within 15 ms, and were observable for approximately 2 min at pH 7.4 but for less than 5 s at pH 4.2. The first-order rate constant for the decay of the intermediate has been determined by stopped-flow spectroscopy, showing the isotope effect, k(H)/k(D) of 1.4 in D(2)O. The intermediate was found to decay mainly from the protonated form by analyzing pH dependences. The enthalpy and entropy of activation suggested that a considerable structure change takes place around the active site during the decay of the intermediate. The EPR spectra at cryogenic temperatures (<27 K) showed two broad signals with g approximately 1.8 and 1.6 depending on pH. We propose an oxygen-centered radical in magnetic interaction with the oxidized type III copper ions as the structure of the three-electron reduced form of dioxygen.     

Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.