Mechanisms of oxidant generation by catalase

Abstract

The enzyme catalase converts solar radiation into reactive oxidant species (ROS). In this study, we report that several bacterial catalases (hydroperoxidases, HP), including Escherichia coli HP-I and HP-II also generate reactive oxidants in response to ultraviolet B light (UVB). HP-I and HP-II are identical except for the presence of NADPH. We found that only one of the catalases, HPI, produces oxidants in response to UVB light, indicating a potential role for the nucleotide in ROS production. This prompts us to speculate that NADPH may act as a cofactor regulating ROS generation by mammalian catalases. Structural analysis of the NADPH domains of several mammalian catalases revealed that the nucleotide is bound in a constrained conformation and that UVB irradiation induces NADPH oxidation and positional changes. Biochemical and kinetic analysis indicate that ROS formation by the enzyme is enhanced by oxidation of the cofactor. Conformational changes following absorption of UVB light by catalase NADPH have the potential to facilitate ROS production by the enzyme.

Figure 1

Differential effects of UVB on NADPH binding (HP I) and NADPH-independent (HP II) E-coli catalases. Recombinant catalases [(catalase (2.2 μM)] were incubated with 50 μM 2′,7′-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR) and the reaction mixtures were irradiated in uncovered 96-well tissue culture plates (Costar, Corning, NY) with UVB light emitted from two Westinghouse FS20 light tubes. The UVB lights were calibrated with an IL 442A Phototherapy Radiometer (International Light, Newburyport, MA). Fluorescence was quantified using an HTS 7000 plus bio-assay reader (PerkinElmer Life Sciences, Beaconsfield Buckinghamshire, UK) with 495 nm excitation and 520 nm emission filters. E. coli hydroperoxidases were generously provided by Peter Loewen, U. Manotoba).

Figure 2

Model of proposed heme-heme and heme-NADPH linkage in human catalase. (Upper panel) Model illustrating functional dimmer in 1.5A structure of human catalase. (Lower panel) Position of migrating potential tyrosinate radical in adjacent protein domains. We speculate that the migration of this radical facilitates the formation of an electronic charge relay linkage associating two heme moieties in each functional dimer.^,,,,

Figure 3

Detail of potential charge relay network in one catalase tertromer. The red arrow identifies the proposed path of charge relay. Mutations to residues noted have inhibited the rate of peroxide degradation in mammalian and bacterial catalases and enhanced ROS generation in response to UVB light (data not shown).^,,,,