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The peroxidase activity of cytochrome c ‐550 from Paracoccus versutus

2001; Wiley; Volume: 268; Issue: 15 Linguagem: Inglês

10.1046/j.1432-1327.2001.02335.x

1432-1033

Rutger E. M. Diederix, Marcellus Ubbink, Gerard W. Canters,

Photoreceptor and optogenetics research

Next to their natural electron transport capacities, c ‐type cytochromes possess low peroxidase and cytochrome P‐450 activities in the presence of hydrogen peroxide. These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics. This study reports on the peroxidase activity of cytochrome c‐ 550 from Paracoccus versutus and the loss of this activity in presence of H 2 O 2 . The rate‐determining step in the peroxidase reaction of cytochrome c‐ 550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron. The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H 2 O 2 , and the lack of a base catalyst such as the distal His of the peroxidases. This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 m H 2 O 2 . Steady‐state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build‐up of amino‐acid radicals on the protein. The inactivation kinetics in the absence of reducing substrate are mono‐exponential and shown to be concurrent with haem degradation up to 25 m m H 2 O 2 (pH 8.0). At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H 2 O 2 , involving the formation of superoxide and ferrocytochrome c‐ 550.