Journal of Biological Chemistry
Volume 271, Issue 42, 18 October 1996, Pages 26018-26025
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Enzymology
Stimulation by Nitroxides of Catalase-like Activity of Hemeproteins: KINETICS AND MECHANISM*

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The ability of stable nitroxide radicals to detoxify hypervalent heme proteins such as ferrylmyoglobin (MbFeIV) produced in the reaction of metmyoglobin (MbFeIII) and H2O2 was evaluated by monitoring O2 evolution, H2O2 depletion, and redox changes of the heme prosthetic group. The rate of H2O2 depletion and O2 evolution catalyzed by MbFeIII was enhanced by stable nitroxides such as 4-OH-2,2,6,6-tetramethyl-piperidinoxyl (TPL) in a catalytic fashion. The reduction of MbFeIV to MbFeIII was the rate-limiting step. Excess TPL over MbFeIII enhanced catalase-like activity more than 4-fold. During dismutation of H2O2, [TPL] and [MbFeIV] remained constant. NADH caused: (a) inhibition of H2O2 decay; (b) progressive reduction of TPL to its respective hydroxylamine TPL-H; and (c) arrest/inhibition of oxygen evolution or elicit consumption of O2. Following depletion of NADH the evolution of O2 resumed, and the initial concentration of TPL was restored. Kinetic analysis showed that two distinct forms of MbFeIV might be involved in the process. In summary, by shuttling between two oxidation states, namely nitroxide and oxoammonium cation, stable nitroxides enhance the catalase mimic activity of MbFeIII, thus facilitating H2O2 dismutation accompanied by O2 evolution and providing protection against hypervalent heme proteins.

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This work was supported in part by the Szold Foundation, Jerusalem and by Grant 309/931 from the Israel Science Foundation of the Israel Academy of Science, Jerusalem, Israel. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.