Catalase Oxidation Mechanisms 259 



with it, leaving N* and the one-electron oxidation is completed by migration of an 

 electron from carbon. The oxygen finally is protonated to become OH. The metal 

 atom then oxidizes the OH group bound through the hydrogen bond to the a carbon 

 atom. The oxidized OH finally attacks the a carbon atom completing the two- 

 electron oxidation. The resulting carbinolamine spontaneously rearranges to yield 

 the products. It will be appreciated that the original metal complex with the vacant 

 site and adjacent OH group is regenerated in the reaction. 

 M ARGOLiASH '. Some of the observations made during the study of the irreversible inhibition 

 of catalase bear on the points that have just been made. We studied the minimal 

 structural requirements of compounds that showed the aminotriazole type of irre- 

 versible inhibition of catalase. Among other features was an absolute requirement 

 for a free primary amino group. Any substitution on this amino group resulted in a 

 complete disappearance of the inhibitory activity. The second point to consider is 

 that when the haem was separated from the protein and the protein denatured, as 

 occurs with the usual acid-acetone treatment, the irreversible inhibitor remained 

 entirely bound to the protein. The inhibitor must, however, have interferred with the 

 haem in some way since catalase irreversibly inhibited with aminotriazole did not 

 react with the usual ferric ligands such as cyanide or azide. Finally, if one compares 

 the spectrum of irreversibly inhibited catalase with that of catalase-hydrogen peroxide 

 complexes, it seems to be more similar to that of complex I than to any of the others. 

 These various observations led to the idea that the irreversible inhibitors may possibly 

 be covalently bonded to the protein through an amide link to a particular carboxyl 

 group at the active site of the enzyme. This hypothesis is being tested. No direct 

 proof has been obtained as yet, partly because of the obvious experimental difficulties 

 of working with a protein having a molecular weight of 240,000. The similarity of the 

 spectrum of irreversibly inhibited catalase to that of complex I might be due to the 

 binding of the inhibitor to the protein in a manner not entirely dissimilar to the effect 

 of the 'peroxide' in complex I on the active site of the enzyme protein. 



