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1.
Biochemistry ; 47(27): 7108-15, 2008 Jul 08.
Article in English | MEDLINE | ID: mdl-18553939

ABSTRACT

Octopus vulgaris hemocyanin ( Ov-Hc) and one of its minimal functional units ( Ov-g) have been purified, and their spectroscopic features and monooxygenase (phenolase) activity have been examined in detail. The oxy forms of both Ov-Hc and Ov-g are stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea at 25 degrees C; approximately 90 and approximately 75% of the (mu-eta (2):eta (2)-peroxo)dicopper(II) species of Ov-Hc and Ov-g, respectively, remained unchanged after argon (Ar) gas flushing of the sample solutions for 1 h. The catalytic activity of Ov-g in the oxygenation reaction (multiturnover reaction) of 4-methylphenol ( p-cresol) to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of Ov-Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxyhemocyanin proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase. The effect of urea on the redox functions of oxyhemocyanin is discussed on the basis of the spectroscopic analysis and reactivity studies.


Subject(s)
Hemocyanins/metabolism , Mixed Function Oxygenases/metabolism , Octopodiformes/enzymology , Aerobiosis , Anaerobiosis , Animals , Binding Sites , Catalysis , Catechols/chemistry , Catechols/metabolism , Circular Dichroism , Hemocyanins/chemistry , Mixed Function Oxygenases/chemistry , Phenols/chemistry , Phenols/metabolism , Protein Structure, Quaternary , Substrate Specificity , Time Factors
2.
J Am Chem Soc ; 128(21): 6788-9, 2006 May 31.
Article in English | MEDLINE | ID: mdl-16719449

ABSTRACT

Oxygenation of a series of p-substituted phenols to the corresponding catechols (phenolase activity) by the (mu-eta2:eta2-peroxo)dicopper(II) species of Octopus hemocyanin has been directly examined for the first time by using a UV-vis spectroscopic method in a 0.5 M borate buffer solution containing 8 M urea under anaerobic conditions. Preliminary kinetic studies have indicated that the reaction involves an electrophilic aromatic substitution mechanism as in the case of phenolase reaction of tyrosinase. The oxygenation of phenols by hemocyanin also proceeded catalytically when the reaction was carried out under aerobic conditions.


Subject(s)
Hemocyanins/chemistry , Hemocyanins/metabolism , Urea/chemistry , Animals , Cresols/chemistry , Linear Models , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Octopodiformes , Oxygen/metabolism , Spectrophotometry, Ultraviolet
3.
Biochemistry ; 43(36): 11546-53, 2004 Sep 14.
Article in English | MEDLINE | ID: mdl-15350140

ABSTRACT

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.


Subject(s)
Agaricus/enzymology , Catalase/metabolism , Fungal Proteins/metabolism , Mixed Function Oxygenases/metabolism , Monophenol Monooxygenase/metabolism , Animals , Catalase/chemistry , Catalysis , Fungal Proteins/chemistry , Hemocyanins/chemistry , Hemocyanins/metabolism , Hydrogen Peroxide/metabolism , Kinetics , Mixed Function Oxygenases/chemistry , Models, Chemical , Mollusca/enzymology , Monophenol Monooxygenase/chemistry , Oxygen/metabolism , Spectrophotometry, Ultraviolet , Substrate Specificity
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