We have purified to homogeneity a catalase found in extracts of Neurospora crassa 5297a that had been induced for nitrate reductase in a nitrate-supplemented medium. The activity is stabilized in extracts by the protease inhibitor phenylmethanesulfonyl fluoride. The enzyme has an apparent molecular weight of 3.2 × 105 and is composed of four subunits of 8 × 104 molecular weight. The catalytic constant for H2O2 dismutation, at pH 6.8 and 25 °C, is 4.6 × 106 M-1 s-1, and the activation energy is 7.2 ± 0.4 kcal/mol. The protein exhibits no peroxidase activity with guiacol as the substrate. The enzyme is not reducible by sodium dithionite. The inhibitory effect of KCN, NaN3, NaF, Na2SO3, KNO2, and KNO3 on enzyme activity at pH 7 was studied; cyanide and azide were found to be strong inhibitors of activity. Though the protein is homogeneous according to ultracentrifugation and electrophoresis, the iron content averaged 3.4 atoms of Fe/molecule, suggesting that, in common with bovine liver catalase, the isolated protein does not carry a full complement of heme groups. The electronic spectrum exhibits maxima at 280 (1.1 × 105), 400 (8.2 × 104), 590 (1.7 × 104), and 712 nm (3.8 × 103) [absorbancy per mole of iron is given in parentheses]. The inducibility of the enzyme in the presence of nitrate, its molecular weight, and its electronic spectrum all distinguish this catalase from previously reported catalases. Antibodies raised to homogeneous samples of the enzyme were used in Ouchterlony and double-antibody radioimmunoassays to indicate that a minimum of 75% of the catalase induced in the presence of nitrate is identical with the newly identified catalase. Thus, the presence of nitrate in growth media for N. crassa initiates a series of events including synthesis of nitrate reductase, synthesis of catalase apoprotein, and probably the synthesis of the enzymes responsible for the production of the prosthetic group. © 1979, American Chemical Society. All rights reserved.
