The nature of the primary complex of catalase.

There has been considerable discussion on the nature of the chemical configuration of the primary peroxide complexes of the catalases and peroxidases. The simplest hypothesis (l-5)) namely that peroxide complexes are formed in the reaction, is subject to alternative interpretations. For example, it has been postulated that the spectroscopically identifiable reaction product is not a peroxide complex but a compound of the enzyme which involves a higher oxidation state of the iron and which does not contain the components of the peroxide molecule (6). A number of experimental approaches have been employed to determine whether an intermediate precedes the formation of the green primary compound, as must be the case if the oxidation of the enzyme to a higher valency state has occurred. However, no measurable induction period prior to the formation of Complex I has been found 1 millisecond after initiation of the reaction between the catalase (or peroxidase) and peroxide. Both kinetic analysis and spectrophotometric studies failed to disclose the existence of other intermediates apart from Compound I (3, 4). To obtain data at times much shorter than 1 millisecond, the over-all catalase reaction was investigated at high peroxide concentration (10 M). Such experiments showed that if any intermediate were formed preceding the green compound, its lifetime was shorter than IO-* set (7, 8). In the previous studies of bacterial catalase peroxide complexes (Q), dissociation constants could not be determined. Recently, increases in the sensitivity of the spectrophotometric technique made it possible to measure catalase concentrations of the order of several nanomolar (nM) (10). We report here the measurement of the dissociation constant of Complex I of bacterial catalase and methyl hydrogen peroxide. The existence of the dissociation constant defines the type of reaction involved in the formation of the primary complexes of hydroperoxidases, and confirms the possibility of the peroxide enzyme structure. These results limit the concept of the higher oxidation state of iron (6) to a charge transfer complex containing the component, or components, of the peroxide molecule.