Catalases (H202:H202 oxidoreductase, EC 1.11.1.6) from many species are known to be tetramers of 60,000-dalton subunits, with four heme groups per tetramer. Previous authors have determined the amino acid sequence and three-dimensional structure of bovine liver catalase. Studies of the regulation of the pentose phosphate pathway led the present authors to a search for proteins that bind NADP+ and NADPH in human erythrocytes. An unexpected result of that search was the finding that a major reservoir of bound NADPH in human erythrocytes is catalase. Each tetrameric molecule of human or bovine catalase contains four molecules of tightly bound NADPH. The binding sites have the relative affinities NADPH > NADH > NADP+ > NAD+. NADPH does not seem to be essential for the enzymic conversion of H202 to 02 and water but does provide protection of catalase against inactivation by H202.In the presence of catalase (H202:H202 oxidoreductase, EC 1.11.1.6) hydrogen peroxide is rapidly converted to oxygen and water. Catalase was the source of some of the earliest information about the nature of enzymes. Noting the inhibition of catalase by cyanide, Warburg suggested in 1923 that catalase contains iron (1). Chance obtained evidence for an enzyme-substrate complex from studies of the absorption spectrum of catalase under conditions of rapid flow (2). In 1937, Sumner and Dounce crystallized catalase from bovine liver, achieving one of the first successful crystallizations of an intracellular enzyme (3). The complete amino acid sequence of bovine liver catalase is now known (4), and the three-dimensional structure has been determined at resolutions of 2.5 A for the enzyme from bovine liver (5) and 3.5 A for catalase from Penicillium vitale (6). Catalases from different sources exhibit similarities in molecular weight, number of subunits, and types of prosthetic groups (7,8). The enzyme is a tetramer with a total molecular weight of approximately 240,000. Each tetrameric molecule contains four heme groups in which the iron is in the ferric state.We now report that bovine and human catalase also contain four tightly bound molecules of NADPH. This reduced dinucleotide is not essential for activity of catalase. Instead, NADPH decreases the susceptibility of catalase to inactivation when the enzyme is exposed to low concentrations of its toxic substate, H202. Purified samples of human and bovine catalase were found to bind and release NADPH in a manner suggesting that catalase may also function as a regulatory protein, releasing NADP+ when the cell is under peroxidative stress. This release would augment removal of H202 by the glutathione reductase-glutathione peroxidase mechanisms.MATERIALS AND METHODS Assays. Activity of catalase was expressed as the first-order kinetic constant for disappearance of H202, as determined at a wavelength of 240 nm with a recording spectrophotometer (9). Activities of catalase and NADPH diaphorase (10) (NADPH:methylene blue oxidoreductase, EC 1.6.99.1) were measured at 25°C. Assays for NADP and NAD...

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NADPH is known to be tightly bound to mammalian catalase and to offset the ability of the substrate of catalase (H 2 O 2 ) to convert the enzyme to an inactive state (compound II). In the process, the bound NADPH becomes NADP ؉ and is replaced by another molecule of NADPH. This protection is believed to occur through electron tunneling between NADPH on the surface of the catalase and the heme group within the enzyme. The present study provided additional support for the concept of an intermediate state of catalase, through which NADPH serves to prevent the formation (rather than increase the removal) of compound II. In contrast, the superoxide radical seemed to bypass the intermediate state since NADPH had very little ability to prevent the superoxide radical from converting catalase to compound II. Moreover, the rate of NADPH oxidation was several times the rate of compound II formation (in the absence of NADPH) under a variety of conditions. Very little NADPH oxidation occurred when NADPH was exposed to catalase, H 2 O 2 , or the superoxide radical separately. That the ratio exceeds 1 suggests that NADPH may protect catalase from oxidative damage through actions broader than merely preventing the formation of compound II.