BRITTON CHANCE 



the rate of complex I appearance is very little affected by the 

 bromiridate concentration. Apparently an intermediate com- 

 pound of a half life of about 10 sec. at 25 ° C. is formed. Fur- 

 thermore, this intermediate does not transfer oxidizing equiva- 

 lents solely to the peroxidase heme to form complex I; over 

 twice the theoretical amount of bromiridate is required to form 

 one mole of complex I (>4K2lrBr6 complex I) even under 

 optimal reaction conditions. 



A possible explanation of this peculiar reaction is afforded 

 by the observation that these strong oxidizing agents react with 

 various amino acids; Fergusson has found in particular that 

 bromiridate reacts with tyrosine alone as rapidly as it reacts with 

 peroxidase which contains tyrosine (49). 



It is illuminating to compare the action of bromiridate on 

 the peroxidase system to a coupled oxidation reaction in which 

 peroxide is produced from oxygen. Coupled oxidation re- 

 actions can occur by the addition of a separate enzyme system 

 (glucose plus notatin) or by peroxidase itself reacting with 

 dihydroxymaleic acid. In the latter case peroxidase serves as 

 catalyst for both reactions (16). It is not at all unreasonable 

 that a related effect occurs with bromiridate. 



Such a reaction mechanism appears to differ considerably 

 from that obtained with hydrogen peroxide in which the kinetic 

 and titration data indicate that peroxide combines with the iron 

 porphyrin group in a 1 : 1 stoichiometric ratio and in a second- 

 order reaction over a wide range of times. It is clear that the 

 term "substrate" is suitable for peroxide, while bromiridate is 

 surely not a substrate for the direct formation of complex I. 

 On the other hand, bromiridate could be considered to be a 

 substrate for a reaction with peroxidase protein. 



Multi-Enzyme Reaction Sequences 



The classical biochemical approach to the problem of es- 

 tablishing sequences of enzyme reactions has often been the 

 isolation and purification of the enzyme and a reconstruction of 

 the entire sequence in vitro. An excellent example is the recon- 



318 



