ENZYME-SUBSTRATE COMPOUNDS 



to complexes I or II can be formed in the reaction of peroxidase 

 and various strong oxidizing agents other than hydrogen or 

 substituted hydrogen peroxides (37). Although considerable 

 destruction of peroxidase occurs with many agents, and al- 

 though few of these give appreciable enzymatic acitvity, George's 

 results raise interesting questions with regard to the nature and 

 definition of enzyme specificity. If such agents all react with 

 peroxidase by the same mechanism as peroxide, then peroxidase 

 has no absolute specificity for its substrate, any oxidizing agent 

 that can react with the iron-porphyrin structure would seem to 

 be suitable, and the role of the protein in directing the enzyme- 

 substrate combination would seem to be negligible. On the 

 other hand, there is no evidence that the reaction mechanism for 

 these reagents is the same as with peroxide; it is possible that 

 they react with a rather nonspecific end group of the protein and 

 form an intermediate (possibly peroxide) at that site and that 

 this intermediate subsequently interacts with the iron porphyrin 

 group. The kinetic methods described above would seem to be 

 suitable for testing whether or not such an intermediate step 

 exists in the reaction of peroxidase with agents other than perox- 

 ide. Mr. R. R. Fergusson of this laboratory (34) has selected 

 for this test a one-electron oxidant of known and relatively 

 stable properties, potassium bromiridate, K2lrBr6. This re- 

 agent is preferable to others in which peroxide formation would 

 a priori be suspected. The one-electron oxidation should 

 form complex I by a reversal (equation (8)) of the one-electron 

 steps in which complex I is known to react with donor molecules. 



HRP > complex II > complex I (8) 



In confirmation of George's preliminary results, we find no 

 trace of complex II to be formed prior to complex I in the re- 

 action with bromiridate, and this result supports the view that 

 a two-electron oxidant must be formed from the one-electron 

 oxidizing agent before formation of complex I can occur. 



The kinetics of the formation of complex I upon addition of 

 bromiridate have been recorded spectrophotometrically, and 



317 



