An EPR study of <i>Neurospora</i> tyrosinase

Deinum, Johanna; Lerch, Konrad; Reinhammar, Bengt

doi:10.1016/0014-5793(76)80676-x

Cited by 49 publications

(26 citation statements)

References 15 publications

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“…In the case of the material investigated by Mondovi et al [8], which contained 6-8 Cu/l40000 M,, computer simulations of the EPR spectra indicated an equal amount of type-1 and type-2 copper. These results are in contrast to the ratio of 3 to 1 as found by Deinum et al [13] in a sample of ascorbate oxidase prepared by one of us (A. M.) in 1973. This preparation had 8 Cu/enzyme molecule.…”

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confidence: 93%

Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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1. Ascorbate oxidase has been isolated from the green squash Cucurbitapepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20 000 M, could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 f 0.11140000 MI) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 2. The pure enzyme is characterized by the following optical purity indices: A2so/Ablo = 25 +_ 0.5, A330/A610 = 0.65 & 0.05 and A6I0/A500 = 7.0 & 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M-' em-'.3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, g , = 2.058, g, = 2.036; A , = 5.0 mT, A , = A , = 0.5 mT, for the type-2 (non-blue) copper gli = 2.242, g l = 2.053; All = 19.0 mT, A1 = 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra.4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (%ax = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (l/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm ( A s = 1000 f 50 M-'

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confidence: 93%

Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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“…A more recent determination of the molecular weight of the enzyme obtained from N. crassa shows that the enzyme contains close to two copper ions per polypeptide chain. A careful search at various temperatures showed less than 3 % ESR-detectable copper (Deinum et al, 1976). The reaction of nitric oxide with the native and reduced enzyme did not give rise to any ESR signals comparable to the NO-complexes of hemocyanin and mushroom tyrosinase.…”

Section: Tyrosinasementioning

confidence: 87%

ESR of Copper in Biological Systems

Boas

Pilbrow

Smith

1978

Biological Magnetic Resonance

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“…They are strongly antiferromagnetically coupled as found previously for N . crassa and mushroom tyrosinases (Deinum et al, 1976; Schoot Uiterkamp 8t Mason, 1973). However, the spectrum of the oxy form from S .…”

Section: Discussionmentioning

confidence: 99%

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase

Huber¹,

Lerch²

1988

Biochemistry

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The physiochemical properties of wild type and two mutants of Streptomyces glaucescens tyrosinase are reported. The native enzyme contains two coppers at the active site which are EPR nondetectable. The two coppers react stoichiometrically with one hydrogen peroxide molecule giving rise to oxytyrosinase. Its optical features are similar to those reported earlier for a molluscan hemocyanin. The two mutants in which histidine-62 and -189 were changed to asparagine by site-directed mutagenesis have lost their enzymatic activity and their ability to bind oxygen and contain only one copper ion which is fully EPR detectable. The EPR parameters indicate that the remaining copper is in a tetragonally distorted ligand environment. These data are in agreement with His-62 and His-189 serving as copper ligands in S. glaucescens tyrosinase.

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An EPR study of Neurospora tyrosinase

Cited by 49 publications

References 15 publications

Ascorbate Oxidase from Cucurbita pepo medullosa

Ascorbate Oxidase from Cucurbita pepo medullosa

ESR of Copper in Biological Systems

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase

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