Different bands of catalase activity in zymograms (Cat-la Cat le) appe
ar during Neurospora crassa development and under stress conditions. H
ere we demonstrate that singlet oxygen modifies Cat-la, giving rise to
a sequential shift in electrophoretic mobility, similar to the one ob
served in vivo. Purified Cat-la was modified with singlet oxygen gener
ated from a photosensitization reaction; even when the reaction was se
parated from the enzyme by an air barrier, a condition in which only s
inglet oxygen can reach the enzyme by diffusion. Modification of Cat-l
a was hindered when reducing agents or singlet oxygen scavengers were
present in the photosensitization reaction. The sequential modificatio
n of the four monomers gave rise to five active catalase conformers wi
th more acidic isoelectric points. The pi of purified Cat-la-Cat-le de
creased progressively, and a similar shift in pi was observed as Cat-l
a was modified by singlet oxygen. No further change was detected once
Cat-le was reached. Catalase modification was traced to a three-step r
eaction of the heme. The heme of Cat-la gave rise to three additional
heme peaks in a high performance liquid chromatography when modified t
o Cat-1c. Full oxidation to Cat-le 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 th
is is a general feature of the enzyme that could explain in part the v
ariety of catalases seen in several organisms and the modifications ob
served in some catalases, Modification of catalases during development
and under stress could indicate in vivo generation of singlet oxygen.