CATALASE 441 



and Agner {2780) have drawn attention to the somewhat related conversion 

 of peroxidases into oxidases by dihydroxymaleic acid. This has been discussed 

 above. Third, the earHer claim of Keilin and Hartree {IJtSl) that the presence 

 of small amounts of atmospheric oxygen is necessary for the activity of 

 catalase was later abandoned {1497, cf. al.so 1410,2()54>2697,3021). This is 

 not conclusive evidence against Keilin's theory since oxygen required for 

 reaction -2 is formed in reaction 1, but Weiss and Weil-Malherbe {3021) have 

 pointed out that the scheme of Keilin necessitates the assumption of a radical 

 chain mechanism. Unless the unlikely assumption is made that the reduction 

 of oxygen to water in reaction '-2 does not proceed via hydrogen peroxide, the 

 hydrogen peroxide decomposed in reaction 1 is re-formed in reaction ^2. 

 Unless a radical is formed during the reduction of ferricatalase in reaction 1, 

 which initiates a decomposition of peroxide by a radical chain, no destruction 

 of hydrogen peroxide could result. Finally, no other ferrous heme compounds 

 are known to combine with azide except perhaps myeloperoxidase. 



A strong argument in favour of Keilin's theory is the fact that azide and 

 hydroxylamine inhibit the activity of catalase at much smaller concentrations 

 than those at which they combine with ferricatalase {cf. Table II). Keilin 

 explains these results by assuming that azide combines much more strongly 

 with ferrocatalase. Were it not for the possibility that azide may also com- 

 bine or react with the protein part of catalase without visible spectroscopic 

 alteration, these observations would indeed prove that the reaction of 

 uninhibited catalase also proceeds over the ferrous state. In Keilin's experi- 

 ments combination of azide or hydroxylamine with catalase was measured by 

 alteration of the absorption spectrum. Such measurements do not give us 

 any information about reactions which may occur between the catalase 

 protein and azide, nor about more complex reactions occurring only in the 

 presence of hydrogen peroxide. 



Lemberg and Foulkes {1609) have studied the azide inhibition by the 

 oxidimetric method. They found that, unlike the cyanide inhibition, the 

 azide inhibition develops only gradually; for technical reasons this is not 

 revealed in manometric experiments. Since this inhibition can be largely 

 reversed by dilution, its gradual appearance cannot be due to irreversible 

 destruction as in the instances mentioned in Section '2.4. According to 

 Keilin's theory one might assume that it is caused by a gradually established 

 equilibrium involving the reduction of the catalase iron. Against this, how- 

 ever, speaks, first, the observation that oxygenation after disappearance of 

 the hydrogen peroxide did not reactivate the enzyme, and, second, that 

 after catalase in the presence of carbon monoxide and azide had been com- 

 pletely inactivated, reoxygenation caused no reappearance of the enzyme 

 activity {1(>99) : according to Keilin the ferrous azide catalase, with or without 

 carbon monoxide, is very autoxidizable. These findings do not appear to be 

 in agreement with Keilin's theory.* 



* Some recent ob.servations (Lemberg and Foulkes, 1698a) suggest that the "ferrous 

 azide catalase" and "ferrous hydroxylamine catalase" may be nitric oxide ferrocatalase, 

 the nitric oxide being produced by an enzyme-catalyzed ozidation of azide or hydroxyl- 

 amine. 



