Bengt Reinhammar

Oxidation-reduction potentials of the electron acceptors in laccases and stellacyanin

1. Laccase (p-diphenol: O2 oxidoreductase, EC 1.10.3.2) obtained from the fungus Polyporus versicolor and laccase and stellacyanin from the lacquer tree Rhus vernicifera were titrated with reducing and oxidizing agents in an anaerobic optical cell equipped with a combined metal electrode for simultaneous optical and potentiometric titrations of the chromophores absorbing at about 610 nm in all three proteins and the 330-nm chromophores in the laccases. 2. The oxidation-reduction potential of the “blue” copper atom in stellacyanin was found to be 184 mV when the protein was dissolved in a sodium phosphate buffer, pH 7.1 and ionic strength 0.3. 3. The oxidation-reduction potential of Type 1 copper and the two-electron acceptor, connected with the 330-nm chromophore, in Rhus laccase was found to be dependent on the concentration of hexacyanoferrate which was used as an electron mediator in the titrations. With about three times excess of this substance, compared to the enzyme concentration, the oxidation-reduction potentials of Type 1 copper and the two-electron acceptor were 434 mV and 483 mV, respectively, when the enzyme was dissolved in sodium phosphate buffer, pH 7.5 and ionic strength 0.2. If the concentration of hexacyanoferrate was only about one third the concentration of enzyme, under otherwise similar conditions as above, the oxidation-reduction potentials of Type 1 copper and the two-electron acceptor decreased to 394 mV and 434 mV, respectively. The increase in oxidation-reduction potential of the Type 1 copper and the two-electron acceptor in the presence of excess hexacyanoferrate seems to be due to a specific reduction of Type 2 copper by the hexacyanoferrate (II). 4. For Polyporus laccase the oxidation-reduction potential of Type 1 copper was extremely high. A value of 785 mV was obtained for an enzyme solution in sodium phosphate buffer, pH 5.5 and ionic strength 0.2, which also contained small amounts of some metal cyanides as electron mediators. The 330-nm chromophore was found to be a two-electron acceptor with an oxidation-reduction potential of 782 mV under these experimental conditions. In the presence of excess of the electron-mediating metal cyanides or with about 100 times excess of the inhibitor NaF the oxidation-reduction potential of Type 1 copper was about the same as above. The potential of the two-electron acceptor decreased to 570 mV, however, when fluoride was present.

Purification and Properties of Laccase and Stellacyanin from Rhus vernicifera

Abstract 1. 1. A new simple and efficient method for the preparation of laccase ( p -diphenol:oxygen oxidoreductase, EC 1.10.3.2) and stellacyanin from the latex of the Japanese lacquer tree Rhus vernicifera is presented. The two proteins were obtained in increased yields as compared with earlier methods and in a high degree of purity, as judged from ultracentrifugation, gel electrophoresis, and isoelectric-focusing electrophoresis as well as from chemical and spectroscopic properties. Together with the main components of laccase and stellacyanin, other chromatographic forms of both proteins were always obtained in small amounts. 2. 2. The amino acid and elemental compositions of laccase were determined. Only about 55% of the laccase molecule is made up of amino acid residues, which accounts for the low nitrogen content of 10.4%. The copper content of laccase is 0.23%, which corresponds to four copper atoms per protein molecule of a molecular weight of 110000.

The state of copper in stellacyanin and laccase from the lacquer tree Rhus vernicifera

1. 1. The blue protein (stellacyanin) and the laccase (p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) obtained from the latex of the Japanese lacquer tree Rhus vernicifera have been studied by electron paramagnetic resonance (EPR) and optical spectroscopy. 2. 2. For stellacyanin the EPR parameters have been obtained with results differing from earlier published values. Above pH 9 the spectral properties of this protein are slightly changed, but in the whole pH range 1–11.5 the Cu2+ has Type 1 character, i.e. it has a strong blue color and a narrow copper hyperfine structure. These unusual features disappear concomitantly and irreversibly at still higher pH. 3. 3. The total EPR intensity of the laccase accounts for 50% of the copper content only. The spectrum consists of two components of equal intensity. It is inferred that each molecule contains four copper ions, one Type 1 Cu2+, which in reduction experiments is selectively reduced, one “non-blue” Type 2 Cu2+ with a normal EPR spectrum, and two EPR-nondetectable copper ions. 4. 4. As with fungal laccase and ceruloplasmin the Type 2 Cu2+ is a preferred binding site of inhibiting anions. At pH 10–12 the Type 1 Cu2+ has slightly different spectral properties, but it still has Type 1 character. 5. 5. It is suggested that the two copper ions in Rhus laccase not seen in EPR are associated with the optical absorption band at 330 nm and that these ions are essential for the function of the enzyme.

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

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.

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., 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).

The mechanism of electron transfer in laccase-catalysed reactions

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.

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.

EPR studies on the anaerobic reduction of fungal laccase. Evidence for participation of type 2 copper in the reduction mechanism.

1. In anaerobic reduction studies on fungal laccase B (p-diphenol:O2 oxidoreductase, EC 1.14.18.1) with the EPR and stopped-flow techniques it was found that the type 2 copper of the enzyme is rapidly undergoing a reduction-oxidation cycle which is followed by a slower reduction in a couple of seconds. An intermediate EPR signal of unknown origin is formed in the same time-range as the initial reduction of type 2 copper and disappears again when this copper ion is reoxidized. 2. The rate of the anaerobic reoxidation of type 2 copper is similar to the reduction rate of the two-electron acceptor, suggesting that they are interacting in the electron transfer of the enzyme. 3. The changes in the reaction rates of both type 2 and type 3 copper appear to be affected in a similar way by changes in pH. 4. The EPR signal of the type 2 Cu2+ suggests that this ion is liganded to one or more nitrogens.

The state of copper in Neurospora laccase

1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined. 2. The molecular weight as determined by gel filtration in 6 M guanidine hydrochloride and by sodium dodecyl sulfate gel electrophoresis is found to be 64 000. The enzyme contains 3.8 copper ions per 64 000. 3. The visible and the near ultraviolet difference absorption spectrum shows two maxima, at 330 and 595 nm, and a shoulder at about 720 nm. The circular dichroism spectrum between 300 and 760 nm contains five bands in the oxidized enzyme. After reduction of the enzyme with ascorbate there remains only a band at 305 nm. 4. EPR measurements show that 52% of the total copper in the protein is paramagnetic. Two EPR signals of equal intensity with different hyperfine splitting constants, of 9 and 18.5 mT, are present, which are assigned to Type 1 Cu2+ and Type 2 Cu2+, respectively, as found in other blue copper-containing oxidases.

The type 2 copper of ascorbate oxidase

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at -30 degrees C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu2+ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu2+ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)6(3), large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.