Ž . Bioelectrochemistry 53 2000 11–16 www.elsevier.comrlocaterbioelechem Electrooxidation of phenol by catalase immobilized on graphite electrodes E. Horozova ) , N. Dimcheva, Z. Jordanova Department of Physical Chemistry, UniÕersity of PloÕdiÕ, 24 Tsar Assen St., PloÕdiÕ 4000, Bulgaria Received 16 August 1999; received in revised form 13 December 1999; accepted 24 July 2000 Abstract Ž . Electrocatalytic properties of catalase CAT immobilized on graphite and soot to mediate electrooxidation of phenol have been investigated. The kinetic parameters — K , k , DG ) and Z of the process studied were calculated. Conclusions on a probable m s Ar 0 mechanism of the biocatalytic and electrochemical process observed were drawn from the calculated values of activation and kinetic parameters. A quantitative UV-spectrophotometrical approach was used as an analytical tool. The electrochemical oxidation of phenol was examined with potentiodynamic and polarization curves’ method. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Mechanism; Electron transfer; Electrooxidation; Phenol; Immobilized catalase; Graphite 1. Introduction Ž . Catalase CAT is regarded as one of the most common enzymes in plant and animal tissues and has a protection function connected with the decomposition of hydrogen peroxide. The CAT macromolecule consists of four sub- units, each of them involving ferriporfyrin as a prosthetic wx group 1 . CAT is a highly specific enzyme and its basic function is high performance catalysis of hydrogen perox- ide decomposition with liberation of water and molecular oxygen. Besides that, CAT also shows a moderate peroxi- dase activity, i.e. it can speed up oxidation reaction with wx hydrogen peroxide 2 . It has been proved by spectroscopy that CAT, like peroxidase forms three compounds, when reacted with hydrogen peroxide. When CAT reacts with Ž hydrogen peroxide, compound I an intermediate . enzyme–substrate complex is formed. Furthermore, it can also oxidize hydrogen peroxide. The loss of an oxidation ) Corresponding author. Tel.: q 359-32-261-552; fax: q 359-32-635- 049. Ž . E-mail address: horozova@argon.acad.bg E. Horozova . equivalent in compound I leads to the formation of com- pound II. Compound III is formed on the oxidation of compound II with hydrogen peroxide and has three oxida- Ž . w x tion equivalents of Fe III 2,3 . Compound I has high activity and takes part in the enzymatic process. Not only hydrogen peroxide, but also other donors can react with compound I, ethanol for example. The activity of com- pound II is lower than that of compound I, while com- wx pound III has no enzymatic activity at all 3 . In biocatalytic and electrochemical systems, CAT is mainly used in immobilized state. The effect of the support used on the thermal stability of immobilized CAT was wx studied 4 . In electrochemical systems, CAT is used to fabricate enzyme electrodes for electrochemical determina- tion of various substrates. CAT biosensors, not only for wx specific determination of hydrogen peroxide 5 but also wx for the inhibitors of the enzyme — fluorides, cyanides 6 , were described. CAT is also used in co-immobilization with H O -producing or consuming enzymes such as glu- 2 2 w x wx w x cose oxidase 7,8 , lactate oxidase 9 , peroxidase 10 , w x w x glutamate oxidase 11,12 , L-lysine-alpha-oxidase 13 or w x choline oxidase 14 . The objective of the present work is to clarify the mechanism and the rate limiting step of the biocatalytic oxidation of phenol by immobilized CAT. 0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. Ž . PII: S0302-4598 00 00089-1