The type 2 copper of ascorbate oxidase |
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| Affiliation: | 1. Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, S-413 90 Göteborg, Sweden;2. Istituto Sperimentale per la Nutrizione Della Piante, I-10125 Torino, Italy;1. Departamento de Ingeniería Eléctrica -SEES, CINVESTAV-IPN, Av IPN 2508, Col Zacatenco, D.F. 07360, Mexico;2. Institute of Molecules & Materials of Le Mans (IMMM) UMR CNRS 6283, Université du Maine, Le Mans, France;3. Institute of Physics, Jan Dlugosz University in Czestochowa, Al.Armii.Krajowej, 13/15, 42-200 Czestochowa, Poland;1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;2. School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, North Fourth Road, Shihezi, Xinjiang 832003, China;1. The University of North Carolina at Chapel Hill, United States;2. Pfizer, United States;1. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland;2. Institut Galien Paris-Sud, UMR 8612, CNRS, Université Paris-Sud, Université Paris-Saclay, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry cedex, France;3. Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada;1. School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, 689-798, Republic of Korea;2. Department of Sociology, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, South Korea;1. Department of Inorganic Chemistry, Faculty of Chemistry, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain;2. Bioarray SL, Alicante, Parque Científico de la UMH, Avda Universidad s/n, 03202 Elche, Alicante, Spain |
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| Abstract: | Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at −30°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°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°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)63−, 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. |
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