Abstract
Different bands of catalase activity in zymograms (Cat-1a-Cat-1e) appear during Neurospora crassa development and under stress conditions. Here we demonstrate that singlet oxygen modifies Cat-1a, giving rise to a sequential shift in electrophoretic mobility, similar to the one observed in vivo. Purified Cat-1a was modified with singlet oxygen generated from a photosensitization reaction; even when the reaction was separated from the enzyme by an air barrier, a condition in which only singlet oxygen can reach the enzyme by diffusion. Modification of Cat-1a was hindered when reducing agents or singlet oxygen scavengers were present in the photosensitization reaction. The sequential modification of the four monomers gave rise to five active catalase conformers with more acidic isoelectric points. The pI of purified Cat-1a-Cat-1e decreased progressively, and a similar shift in pI was observed as Cat-1a was modified by singlet oxygen. No further change was detected once Cat-1e was reached. Catalase modification was traced to a three-step reaction of the heme. The heme of Cat-1a gave rise to three additional heme peaks in a high performance liquid chromatography when modified to Cat-1c. Full oxidation to Cat-1e shifted all peaks into a single one. Absorbance spectra were consistent with an increase in asymmetry as heme was modified. Bacterial, fungal, plant, and animal catalases were all susceptible to modification by singlet oxygen, indicating that this is a general feature of the enzyme that could explain in part the variety of catalases seen in several organisms and the modifications observed in some catalases. Modification of catalases during development and under stress could indicate in vivo generation of singlet oxygen.
Highlights
The electron affinity of O2 makes it a reactive compound
Illumination alone of the sunflower cell extract was sufficient to cause a shift in catalase electrophoretic mobility (Fig. 5H, lanes 1 and 2), but this change in mobility was more prominent in the presence of singlet oxygen. 5-amino salicylic acid (5-ASA) inhibited the sunflower catalase activity but not the Z. mays enzyme (Fig. 5, H and I, lane 8)
Singlet Oxygen Modified Cat-1a—Modification of purified Cat-1a was dependent on O2; no modification occurred under argon
Summary
Vol 273, No 17, Issue of April 24, pp. 10630 –10637, 1998 Printed in U.S.A. (Received for publication, October 31, 1997, and in revised form, February 11, 1998). The hydroxyl radical, one of the most reactive species known, reacts immediately with almost any cellular compound giving rise to alterations, such as modified or broken proteins and nucleic acids [4] It is understandable why catalases are one of the most efficient enzymes known [31]. Since we have demonstrated enzyme inactivation due to ROS-specific oxidation under cell differentiation and stress conditions [24, 25], it was important to determine if antioxidant enzymes such as catalases were vulnerable to in vivo alteration by ROS. We show that Cat-1 was oxidized through a sequential reaction of the four monomers with singlet oxygen, giving rise to active catalase conformers with more acidic isoelectric points. Catalases from different organisms were modified by singlet oxygen, indicating a general feature of the enzyme that could explain in part the variety of catalases seen in several organisms [47,48,49,50,51] and the observed modifications in some catalases [52,53,54,55]
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