Pablo Rangel

Oxidation of catalase by singlet oxygen.

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.

Singlet oxygen is part of a hyperoxidant state generated during spore germination

We show that singlet oxygen is generated in asexual spores (conidia) from Neurospora crassa at the onset of germination. Oxidation of N. crassa catalase-1 (Cat-1) was previously shown to be caused by singlet oxygen (Lledías et al. J. Biol. Chem. 273, 1998). In germinating conidia, increased protein oxidation, decrease of total protein, Cat-1 oxidation and accumulation of cat-1 mRNA was detected. These changes were modulated in vivo by light intensity, an external clean source of singlet oxygen, and by carotene amount and content of coordinated double bonds. Conditions that stimulated singlet oxygen formation increased Cat-1 oxidation and accumulation of cat-1 mRNA. Germinating conidia from mutant strains altered in carotene synthesis showed increased levels of protein degradation, Cat-1 oxidation and accumulation of cat-1 mRNA. During germination Cat-1a was oxidized, oxidized Cat-1c-Cat-1e conformers disappeared and Cat-1a was synthesized de novo. Furthermore, spontaneous oxygen-dependent chemiluminescence increased as soon as conidia absorbed dissolved oxygen. Low-level chemiluminescence is due to photon emission from excited electrons in carbonyls and singlet oxygen as they return to their ground state. H2O2 added to conidia under Ar caused a peak of chemiluminescence and germination of 20% of conidia, suggesting that a hyperoxidant state suffices to start germination under anaerobic conditions. Taken together, these results show that singlet oxygen is part of a hyperoxidant state that develops at the start of germination of conidia, in consonance with our proposal that morphogenetic transitions occur as a response to a hyperoxidant state.

MOLECULAR AND KINETIC STUDY OF CATALASE-1, A DURABLE LARGE CATALASE OF NEUROSPORA CRASSA

Catalase-1 (Cat-1), one of the two monofunctional catalases of Neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. Cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. Like most monofunctional catalases, Cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a pH range of 4-12, and inactivated by 3-amino-1,2,4-triazole. Unlike most monofunctional catalases, Cat-1 consists of 88 kDa monomers that are glycosylated with alpha-glucose and/or alpha-mannose, is unusually stable, and is not inactivated or inhibited by hydrogen peroxide. Cat-1 was more resistant than other catalases to heat inactivation and to high concentrations of salt and denaturants. Cat-1 exhibited unusual kinetics: at molar concentrations of hydrogen peroxide the apparent V was 10 times higher than at millimolar concentrations. Inactivation of Cat-1 activity with azide and hydroxylamine was according to first order kinetics, while cyanide at micromolar concentrations was a reversible competitive inhibitor.

Interactions of calcium with yeast mitochondria

The interactions of Ca2+ with mitochondria from Saccharomyces cerevisiae were explored. Mitochondria were loaded with the metallochromic dye Fluo-3 to measure the concentration of free calcium in the matrix. Addition of EGTA or Ca2+ led to fluctuations in mitochondrial free calcium between 120 and 400 nM. Ca2+ variations were slower at 4 degrees C than at 25 degrees C or in the presence of phosphate instead of acetate. The net uptake of 45Ca2+ was higher with phosphate than with acetate. The optimum pH for Ca2+ uptake was 6.8. Ruthenium red did not affect the uptake of Ca2+. Addition of antimycin-A or uncouplers led to a small and transient release of Ca2+. Addition of EGTA or the monovalent cations Na+ or K+ resulted in higher release of Ca2+. Site I but not site II dependent O2 consumption was partially inhibited by EGTA. The effect of Ca2+ on NADH oxidation is similar to results reported with enzymes from mammalian sources which use NADH, such as the pyruvate, isocitrate and oxoglutarate dehydrogenases.

Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A

Abstract In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa3-OII and aa3-II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type – strain PYM1 contained the membrane-bound subunits caa3-COI and aa3-I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo.

Chapter 15 Cell differentiation as a response to oxidative stress

Abstract Photosynthetic dioxygen (O 2 ) discharge and accumulation in the atmosphere compelled cells to adapt or to die in the presence of a reactive compound and to protect themselves from it and from the inevitable formation of more reactive oxygen species (ROS). At the same time, by using O 2 as final electron acceptor, early heterotrophs could obtain more energy from reduced carbon and attain faster growth. However, fast growing aerobes were made more dependent on the availability of reduced carbon. In the fight for food, microorganisms colonized different niches including other organisms, which they parasitized or ate. With time, life-damaging ROS became signals used by cells to regulate growth and proliferation, cell differentiation and cell death. We have proposed that cell differentiation is a response to oxidative stress. In different eukaryotic microorganisms, the presence of ROS and the induction of antioxidant mechanisms are associated with development. Moreover, the superoxide-producing NADPH-oxidases proved essential for some developmental processes. The small GTPase RAS-1 also regulates the production of ROS and different mutations in the corresponding gene affect cell differentiation. The process of asexual reproduction (conidiation) in Neurospora crassa involves three morphogenetic transitions, each preceded by a hyperoxidant state. An accentuated redox imbalance, the oxidation and degradation of oxidized protein, oxidative inactivation of different enzyme activities, and increased production of ROS characterize the hyperoxidant state. Null mutants in catalase genes show increased levels of development. Strains without superoxide dismutase express a conidiation rhythm, similar to the “band” strain, which contains a semi-dominant ras -1 mutation. Mutation of NADPH-oxidase genes reduced or completely abolished different developmental processes. Together, these results support our view on cell differentiation as a response to oxidative stress.