Catalase Oxidation Mechanisms 



257 



The analysis just described would appear to have little relevance to catalase since 

 no values for a dissociation constant of the primary complex have been reported; the 

 values given are regarded to be 'apparent' dissociation constants (Eq. 7-9), due 

 to endogenous donor. In bacterial catalase the latter is present in negligible amounts 

 and the possibility of accurate titrations presents itself. Such titrations have been 

 carried out with sufficient spectrophotometric sensitivity that millimicromolar amounts 

 of intermediate compounds can be detected. The experimental details are given 

 elsewhere, but a typical result is included in Fig. 1, which gives both/? and p^ plots 

 for the reaction of catalase and methyl hydrogen peroxide. It is clear that the data 



P 

 m/<M 5 



(solid) 



ICOO 

 (e-p)(xo-p){m/<M)' 

 Fig. 1. 



P^ 2 

 50 IrVi/iM) 



(dashed) 



favour the hypothesis that catalase forms a peroxide complex in which the components 

 of the peroxide are retained. 



This result is consistent with our early hypothesis that the interaction of peroxide 

 with catalase was not limited to the iron atom but could include interaction with the 

 methene bridges of the porphyrin ring. At that time a hypothesis was put forward 

 'as to whether the porphyrin ring is actually oxidized on formation of the primary 

 complex by electron transfer from the iron-peroxide complex. . . .' (Chance, /. biol. 

 Clieni. 179, 1331, 1949). This idea also is in accordance with the hypothesis here put 

 forward by Williams and Brill, except that we regard the band in the red as adequately 

 indicative of interaction with the porphyrin. 

 George: I would like to ask Chance whether he has any data on the reaction of horse- 

 radish peroxidase with methyl hydroperoxide, similar to that for catalase, since the 

 behaviour of horseradish peroxidase with many strong oxidizing agents would suggest 

 that the specific structural elements of hydrogen peroxide or ethyl hydroperoxides 

 are not essential in the oxidant for the higher oxidation states to be formed; although 

 of course there is a possibility that OH group attachment could occur through the 

 intervention of a solvent water molecule at a special site on the prosthetic group. 



The reactions in question may be summarized by the following scheme. 



Fe"! y Fe'v > Fe^ 



(Ferripero.xidase) /Complex II \ /Complex I \ 

 \ Compound 11/ \ Compound 1/ 



Hydrogen peroxide and alkyl hydroperoxides, both two-equivalent oxidizing agents, 

 are particularly effective in oxidizing Fe'" -> Fe^. With potassium chloriridate, a 

 one-equivalent oxidizing agent, an excess has to be used because of its additional 

 reactions with oxidizable groups on the protein, and the product formed is Fe^. 



