Tightly bound NADPH in <i>Proteus mirabilis</i> PR catalase

Abstract

Previous work with catalase from Proteus mirabilis PR, a mutant with high resistance to H 2 O 2 , had shown that two different forms of this enzyme, named A and B, could be resolved by anion exchange chromatography. The present results showed that catalase B differed from A by the presence of bound NADPH. Direct evidence for the presence of this dinucleotide within the bacterial catalase was obtained using high performance liquid chromatography. The obtention of pure catalase B without any addition of external NADPH showed that this dinucleotide was bound tightly to the protein as to be carried along with the protein during purification. Catalase complex II was formed from either-enzyme A or B by the continuous supply of low amounts of hydrogen peroxide as in the case of bovine liver catalase. The reversal of complex II formation from catalase B was obtained using a NADPH-regenerating system (isocitrate plus isocitrate dehydrogenase) in the absence of any added dinucleotide, confirming that this cofactor was present in the enzyme molecule. The addition of dinucleotide was required when performing the same experiment with catalase A. This was the first observation of NADPH binding by a bacterial catalase, and the results agreed with the former theory where NADPH is considered as a rescuer of the enzyme from inactivation in the form of complex II.Key words: catalase, bovine liver catalase, NADPH, Proteus mirabilis.