Oxidation of Catalase by Singlet Oxygen
1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês
10.1074/jbc.273.17.10630
ISSN1083-351X
AutoresFernando Lledı́as, Pablo Rangel, Wilhelm Hansberg,
Tópico(s)bioluminescence and chemiluminescence research
ResumoDifferent 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. 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. Photosynthetic evolution of dioxygen into the atmosphere and its subsequent accumulation led to formation of an ozone layer in the stratosphere, which permitted the dispersal of microorganisms around earth by absorbing damaging ultraviolet radiation from the sun (1Schopf J.W. Earth's Earliest Biosphere, Its Origin and Evolution. Princeton University Press, Princeton, NJ1983Google Scholar, 2Kastings J.F. Science. 1993; 259: 920-926Crossref PubMed Scopus (1010) Google Scholar). Atmospheric dioxygen also led to the evolution of adaptation mechanisms to live with a poisonous gas (3Fridovich I. BioScience. 1977; 27: 462-466Crossref Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar).The electron affinity of O2 makes it a reactive compound. Furthermore, dioxygen generates more reactive intermediates in its sequential univalent reduction to water. Likewise, singlet oxygen, excited states of O2, are highly reactive species. They arise upon absorption of radiation by O2, either directly or through prevalent cellular compounds such as tetrapyrrols, flavins, pterins, chlorophylls, and retinoids. Reactive oxygen species (ROS) 1The abbreviation used are: ROS, reactive oxygen species; PB, phosphate buffer; PAGE, polyacrylamide gel electrophoresis; HPLC, high- performance liquid chromatography; Me2SO, dimethyl sulfoxide; 5-ASA, 5-acetyl salicilic acid. 1The abbreviation used are: ROS, reactive oxygen species; PB, phosphate buffer; PAGE, polyacrylamide gel electrophoresis; HPLC, high- performance liquid chromatography; Me2SO, dimethyl sulfoxide; 5-ASA, 5-acetyl salicilic acid. are inevitably produced in cells under aerobic or microaerobic conditions (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar).Primeval cells, which originated in an anoxic environment (2Kastings J.F. Science. 1993; 259: 920-926Crossref PubMed Scopus (1010) Google Scholar), had either to hide from O2, or to evolve mechanisms for efficient reduction of entering O2, disposal of ROS, and sequestering of transition metals, which participate in reducing O2 into ROS. Respiration gave early aerobic heterotrophs a new source of energy, but more than anaerobes made them depend on the availability of reduced carbon, indispensable for O2reduction. Thus, since the generation of an oxidant atmosphere, the threat of damaging effects caused by ROS has been prevalent for all organisms. This threat was further utilized by some species that developed devices for the controlled production of ROS, as seen in many host-parasite relationships (5Daub M.E. Leisman G.B. Clark R.A. Bowden E.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9588-9592Crossref PubMed Scopus (64) Google Scholar, 6Levine A. Tenhaken R. Dixon R. Lamb C. Cell. 1994; 79: 583-593Abstract Full Text PDF PubMed Scopus (2250) Google Scholar, 7Badway J.A. Karnovsky M.L. Annu. Rev. Biochem. 1980; 49: 695-726Crossref PubMed Scopus (840) Google Scholar).Because dioxygen and its unavoidable ROS have been of such importance for survival, cells developed mechanisms to exquisitely detect the presence of O2 and ROS for regulation of metabolism and antioxidant responses (8Farr S.B. Kogoma T. Microbiol. Rev. 1991; 55: 561-585Crossref PubMed Google Scholar, 9Zitomer R.S. Lowry C.V. Microbiol. Rev. 1992; 56: 1-11Crossref PubMed Google Scholar, 10Luchi S. Lin E.C.C. Mol. Microbiol. 1993; 9: 9-15Crossref PubMed Scopus (125) Google Scholar, 11Allen J.F. FEBS Lett. 1993; 322: 203-207Crossref Scopus (96) Google Scholar, 12Kullik I. Storz G. Redox Rep. 1994; 1: 23-29Crossref PubMed Google Scholar, 13Eisenstark A. Calcutt M. Becker-Hapak M. Ivanova A. Free Radic. Biol. Med. 1996; 21: 975-993Crossref PubMed Scopus (102) Google Scholar). Most interestingly, ROS became signals used by cells to regulate growth or proliferation (14Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1057) Google Scholar), cell differentiation (15Hansberg W. Aguirre J. J. Theor. Biol. 1990; 142: 201-221Crossref PubMed Scopus (162) Google Scholar, 16Hansberg W. Cienc. Cult. 1996; 48: 67-74Google Scholar, 17Hansberg W. de Groot H. Sies H. Free Radic. Biol. Med. 1994; 14: 287-293Crossref Scopus (111) Google Scholar, 18Allen R.G. Farmer K.J. Toy P.L. Newton R.K. Sohal R.S. Nations C. Dev. Growth Differ. 1985; 27: 615-620Crossref Scopus (20) Google Scholar) and death (14Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1057) Google Scholar, 19Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2093) Google Scholar).ROS are related to the arrest of growth and the start of cell differentiation. We have detected a hyperoxidant state at the start of all three morphogenetic transitions of Neurospora crassaasexual development (conidiation) (15Hansberg W. Aguirre J. J. Theor. Biol. 1990; 142: 201-221Crossref PubMed Scopus (162) Google Scholar, 16Hansberg W. Cienc. Cult. 1996; 48: 67-74Google Scholar, 17Hansberg W. de Groot H. Sies H. Free Radic. Biol. Med. 1994; 14: 287-293Crossref Scopus (111) Google Scholar, 20Toledo I. Hansberg W. Exp. Mycol. 1990; 14: 184-189Crossref Scopus (28) Google Scholar, 21Toledo I. Noronha-Dutra A.A. Hansberg W. J. Bacteriol. 1991; 173: 3243-3249Crossref PubMed Google Scholar, 22Toledo I. Aguirre J. Hansberg W. Microbiology. 1994; 140: 2391-2397Crossref PubMed Scopus (34) Google Scholar, 23Toledo I. Rangel P. Hansberg W. Arch. Biochem. Biophys. 1995; 319: 519-524Crossref PubMed Scopus (40) Google Scholar). Increased generation of ROS leads to specific oxidation of some enzymes and massive protein oxidation and degradation (20Toledo I. Hansberg W. Exp. Mycol. 1990; 14: 184-189Crossref Scopus (28) Google Scholar, 24Aguirre J. Hansberg W. J. Bacteriol. 1986; 166: 1040-1045Crossref PubMed Google Scholar, 25Aguirre J. Rodrı́guez R. Hansberg W. J. Bacteriol. 1989; 171: 6243-6250Crossref PubMed Google Scholar). Specific modifications induced by ROS have been detected in protein that bind Fe(II) directly (24Aguirre J. Hansberg W. J. Bacteriol. 1986; 166: 1040-1045Crossref PubMed Google Scholar, 26Farber J.M. Levine R.L. J. Biol. Chem. 1986; 261: 4574-4578Abstract Full Text PDF PubMed Google Scholar), bind a Fe(II) chelate complex (25Aguirre J. Rodrı́guez R. Hansberg W. J. Bacteriol. 1989; 171: 6243-6250Crossref PubMed Google Scholar), or have a noncatalytic iron-sulfur cluster (27Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar). These modifications inactivate the enzymes and make them more susceptible to endogenous proteolytic activity (27Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar, 28Rivett A.J. J. Biol. Chem. 1987; 260: 300-305Abstract Full Text PDF Google Scholar, 29Davies K.J.A. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 30Roseman J.E. Levine R.L. J. Biol. Chem. 1985; 262: 2101-2110Abstract Full Text PDF Google Scholar).Safe disposal of H2O2 in cells is carried out by catalases and peroxidases. Hydrogen peroxide is formed mainly from dismutation of superoxide, which is generated by O2capturing an electron, usually from electron transport chains. Hydrogen peroxide is also a product of some oxidases. Being uncharged and not very reactive, it can diffuse between cell compartments. Its toxicity is traced to the formation of the hydroxyl radical upon capture of an electron, for instance, from Fe(II) or Cu(I). 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 (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). Thus, it is understandable why catalases are one of the most efficient enzymes known (31Fita I. Rossmann M.G. J. Mol. Biol. 1985; 185: 21-37Crossref PubMed Scopus (355) Google Scholar). It is so efficient that it cannot be saturated by H2O2at any concentration.Catalases are prevalent in most organisms (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). Many microorganisms have more than one catalase, and in some a catalase is related to cell differentiation (32Loewen P.C. Triggs B, L. J. Bacteriol. 1984; 160: 668-675Crossref PubMed Google Scholar, 33Mulvey M.R. Switala J. Borys A. Loewen P.C. J. Bacteriol. 1990; 172: 6713-6720Crossref PubMed Google Scholar, 34von Ossowski I. Mulvey M.R. Leco P.A. Borys A. Loewen P.C. J. Bacteriol. 1991; 173: 514-520Crossref PubMed Scopus (126) Google Scholar, 35Bol D.K. Yasbin R.E. Gene (Amst.). 1991; 109: 31-37Crossref PubMed Scopus (39) Google Scholar, 36Engelmann S. Lindner C. Hecker M. J. Bacteriol. 1995; 177: 5598-5605Crossref PubMed Google Scholar, 37Hartig A. Ruis H. Eur. J. Biochem. 1986; 160: 487-490Crossref PubMed Scopus (73) Google Scholar, 38Cohen G. Rapatz W. Ruis H. Eur. J. Biochem. 1988; 176: 159-163Crossref PubMed Scopus (85) Google Scholar, 39Navarro R.E. Stringer M.A. Hansberg W. Timberlake W.E. Aguirre J. Curr. Genet. 1996; 29: 352-359PubMed Google Scholar, 40Kawasaki L. Wysong D. Diamond R. Aguirre J. J. Bacteriol. 1997; 179: 3284-3292Crossref PubMed Google Scholar, 41Fowler T. Rey M.W. Vaha-Vahe P. Power S.D. Berka R.M. Mol. Microbiol. 1993; 9: 989-998Crossref PubMed Scopus (48) Google Scholar, 42Redinbaugh M.G. Wadsworth G.J. Scandalius J.G. Biochim. Biophys. Acta. 1988; 951: 104-116Crossref PubMed Scopus (101) Google Scholar, 43Willekens H. Langebartels C. Tiré C. van Montagu M. Inzé D. van Camp W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10450-10454Crossref PubMed Scopus (171) Google Scholar, 44Loewen P.C. Switala J. Biochem. Cell Biol. 1988; 66: 707-714Crossref PubMed Scopus (23) Google Scholar, 45Hengge-Aronis R. Cell. 1993; 72: 165-168Abstract Full Text PDF PubMed Scopus (454) Google Scholar). Two catalase genes have been cloned in Aspergillus nidulans, one expressed only for asexual spores (conidia) (39Navarro R.E. Stringer M.A. Hansberg W. Timberlake W.E. Aguirre J. Curr. Genet. 1996; 29: 352-359PubMed Google Scholar, 40Kawasaki L. Wysong D. Diamond R. Aguirre J. J. Bacteriol. 1997; 179: 3284-3292Crossref PubMed Google Scholar). A third catalase activity has been recently found. 2L. Kawasaki and J. Aguirre, personal communication. 2L. Kawasaki and J. Aguirre, personal communication. Neurospora crassa has two 3F. Lledı́as and W. Hansberg, submitted for publication. 3F. Lledı́as and W. Hansberg, submitted for publication. or three (46Chary P. Natvig D.O. J. Bacteriol. 1989; 171: 2646-2652Crossref PubMed Google Scholar) catalase genes. The cat-1 gene mapped to complementation group IIIR has been cloned and partially sequenced (72Baldwin, J. L., Natvig, D. O., Abstracts of the 18th Fungal Genetic Conference, March 21–28, Asilomar, CA, 1995, 31.Google Scholar). It codes for a homotetramer of 320-kDa molecular mass. Cat-1 is present in the whole vegetative life cycle of N. crassa. 3 The cat-2 gene, assigned to complementation group VIIR, has not been cloned. It is expressed under stress conditions and transiently during the formation of conidia.3Catalase-specific activity increases stepwise with each morphogenetic transition of the N. crassa conidiation process.3 Cat-1 was modified in these transitions and under stress conditions.3 Since we have demonstrated enzyme inactivation due to ROS-specific oxidation under cell differentiation and stress conditions (24Aguirre J. Hansberg W. J. Bacteriol. 1986; 166: 1040-1045Crossref PubMed Google Scholar, 25Aguirre J. Rodrı́guez R. Hansberg W. J. Bacteriol. 1989; 171: 6243-6250Crossref PubMed Google Scholar), it was important to determine if antioxidant enzymes such as catalases were also vulnerable to in vivo alteration by ROS. As predicted (15Hansberg W. Aguirre J. J. Theor. Biol. 1990; 142: 201-221Crossref PubMed Scopus (162) Google Scholar, 16Hansberg W. Cienc. Cult. 1996; 48: 67-74Google Scholar), these enzymes are susceptible to oxidation by ROS but not affected in their activity.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. Modification could be traced to a three-step reaction of the heme with singlet oxygen. Catalases from different organisms were similarly modified by singlet oxygen, indicating a general feature of the enzyme that could explain in part the variety of catalases seen in several organisms (47Loewen P.C. Switala J. J. Bacteriol. 1987; 169: 3601-3607Crossref PubMed Google Scholar, 48Kim H-P. Lee J-S. Hah J.Ch. Roe J-H. Micobiology. 1994; 140: 3391-3397PubMed Google Scholar, 49Quail P.H. Scandalios J.G. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1402-1406Crossref PubMed Scopus (48) Google Scholar, 50Eising R. Trelease R.N. Ni W. Arch. Biochem. Biophys. 1990; 278: 258-264Crossref PubMed Scopus (48) Google Scholar, 51Masters C. Pegg M. Crane D. Mol. Cell. Biochem. 1986; 70: 113-120Crossref PubMed Scopus (35) Google Scholar) and the observed modifications in some catalases (52Loewen P.C. Switala J. von Ossowski I. Hillar A. Chistie A. Tattrie B. Nicholls P. Biochemistry. 1993; 32: 10159-10164Crossref PubMed Scopus (62) Google Scholar, 53Murshudov G.N. Grebenko A.I. Barynin V. Dauter Z. Wilson K.S. Vainshtein B.K. Melik-Adamyan W. Bravo J. Ferrán J.M. Ferrer J.C. Switala J. Loewen P.C. Fita I. J. Biol. Chem. 1996; 271: 8863-8868Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 54Buzy A. Bracchi V. Sterjiades R. Chroboczek J. Thibault P. Gagnon J. Jouve H.M. Hurdy-Clergeon G. J. Protein Chem. 1995; 14: 59-72Crossref PubMed Scopus (30) Google Scholar, 55Gouet P. Jouve H-M. Dideberg O. J. Mol. Biol. 1995; 249: 933-954Crossref PubMed Scopus (118) Google Scholar). Photosynthetic evolution of dioxygen into the atmosphere and its subsequent accumulation led to formation of an ozone layer in the stratosphere, which permitted the dispersal of microorganisms around earth by absorbing damaging ultraviolet radiation from the sun (1Schopf J.W. Earth's Earliest Biosphere, Its Origin and Evolution. Princeton University Press, Princeton, NJ1983Google Scholar, 2Kastings J.F. Science. 1993; 259: 920-926Crossref PubMed Scopus (1010) Google Scholar). Atmospheric dioxygen also led to the evolution of adaptation mechanisms to live with a poisonous gas (3Fridovich I. BioScience. 1977; 27: 462-466Crossref Google Scholar, 4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). The electron affinity of O2 makes it a reactive compound. Furthermore, dioxygen generates more reactive intermediates in its sequential univalent reduction to water. Likewise, singlet oxygen, excited states of O2, are highly reactive species. They arise upon absorption of radiation by O2, either directly or through prevalent cellular compounds such as tetrapyrrols, flavins, pterins, chlorophylls, and retinoids. Reactive oxygen species (ROS) 1The abbreviation used are: ROS, reactive oxygen species; PB, phosphate buffer; PAGE, polyacrylamide gel electrophoresis; HPLC, high- performance liquid chromatography; Me2SO, dimethyl sulfoxide; 5-ASA, 5-acetyl salicilic acid. 1The abbreviation used are: ROS, reactive oxygen species; PB, phosphate buffer; PAGE, polyacrylamide gel electrophoresis; HPLC, high- performance liquid chromatography; Me2SO, dimethyl sulfoxide; 5-ASA, 5-acetyl salicilic acid. are inevitably produced in cells under aerobic or microaerobic conditions (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). Primeval cells, which originated in an anoxic environment (2Kastings J.F. Science. 1993; 259: 920-926Crossref PubMed Scopus (1010) Google Scholar), had either to hide from O2, or to evolve mechanisms for efficient reduction of entering O2, disposal of ROS, and sequestering of transition metals, which participate in reducing O2 into ROS. Respiration gave early aerobic heterotrophs a new source of energy, but more than anaerobes made them depend on the availability of reduced carbon, indispensable for O2reduction. Thus, since the generation of an oxidant atmosphere, the threat of damaging effects caused by ROS has been prevalent for all organisms. This threat was further utilized by some species that developed devices for the controlled production of ROS, as seen in many host-parasite relationships (5Daub M.E. Leisman G.B. Clark R.A. Bowden E.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9588-9592Crossref PubMed Scopus (64) Google Scholar, 6Levine A. Tenhaken R. Dixon R. Lamb C. Cell. 1994; 79: 583-593Abstract Full Text PDF PubMed Scopus (2250) Google Scholar, 7Badway J.A. Karnovsky M.L. Annu. Rev. Biochem. 1980; 49: 695-726Crossref PubMed Scopus (840) Google Scholar). Because dioxygen and its unavoidable ROS have been of such importance for survival, cells developed mechanisms to exquisitely detect the presence of O2 and ROS for regulation of metabolism and antioxidant responses (8Farr S.B. Kogoma T. Microbiol. Rev. 1991; 55: 561-585Crossref PubMed Google Scholar, 9Zitomer R.S. Lowry C.V. Microbiol. Rev. 1992; 56: 1-11Crossref PubMed Google Scholar, 10Luchi S. Lin E.C.C. Mol. Microbiol. 1993; 9: 9-15Crossref PubMed Scopus (125) Google Scholar, 11Allen J.F. FEBS Lett. 1993; 322: 203-207Crossref Scopus (96) Google Scholar, 12Kullik I. Storz G. Redox Rep. 1994; 1: 23-29Crossref PubMed Google Scholar, 13Eisenstark A. Calcutt M. Becker-Hapak M. Ivanova A. Free Radic. Biol. Med. 1996; 21: 975-993Crossref PubMed Scopus (102) Google Scholar). Most interestingly, ROS became signals used by cells to regulate growth or proliferation (14Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1057) Google Scholar), cell differentiation (15Hansberg W. Aguirre J. J. Theor. Biol. 1990; 142: 201-221Crossref PubMed Scopus (162) Google Scholar, 16Hansberg W. Cienc. Cult. 1996; 48: 67-74Google Scholar, 17Hansberg W. de Groot H. Sies H. Free Radic. Biol. Med. 1994; 14: 287-293Crossref Scopus (111) Google Scholar, 18Allen R.G. Farmer K.J. Toy P.L. Newton R.K. Sohal R.S. Nations C. Dev. Growth Differ. 1985; 27: 615-620Crossref Scopus (20) Google Scholar) and death (14Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1057) Google Scholar, 19Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2093) Google Scholar). ROS are related to the arrest of growth and the start of cell differentiation. We have detected a hyperoxidant state at the start of all three morphogenetic transitions of Neurospora crassaasexual development (conidiation) (15Hansberg W. Aguirre J. J. Theor. Biol. 1990; 142: 201-221Crossref PubMed Scopus (162) Google Scholar, 16Hansberg W. Cienc. Cult. 1996; 48: 67-74Google Scholar, 17Hansberg W. de Groot H. Sies H. Free Radic. Biol. Med. 1994; 14: 287-293Crossref Scopus (111) Google Scholar, 20Toledo I. Hansberg W. Exp. Mycol. 1990; 14: 184-189Crossref Scopus (28) Google Scholar, 21Toledo I. Noronha-Dutra A.A. Hansberg W. J. Bacteriol. 1991; 173: 3243-3249Crossref PubMed Google Scholar, 22Toledo I. Aguirre J. Hansberg W. Microbiology. 1994; 140: 2391-2397Crossref PubMed Scopus (34) Google Scholar, 23Toledo I. Rangel P. Hansberg W. Arch. Biochem. Biophys. 1995; 319: 519-524Crossref PubMed Scopus (40) Google Scholar). Increased generation of ROS leads to specific oxidation of some enzymes and massive protein oxidation and degradation (20Toledo I. Hansberg W. Exp. Mycol. 1990; 14: 184-189Crossref Scopus (28) Google Scholar, 24Aguirre J. Hansberg W. J. Bacteriol. 1986; 166: 1040-1045Crossref PubMed Google Scholar, 25Aguirre J. Rodrı́guez R. Hansberg W. J. Bacteriol. 1989; 171: 6243-6250Crossref PubMed Google Scholar). Specific modifications induced by ROS have been detected in protein that bind Fe(II) directly (24Aguirre J. Hansberg W. J. Bacteriol. 1986; 166: 1040-1045Crossref PubMed Google Scholar, 26Farber J.M. Levine R.L. J. Biol. Chem. 1986; 261: 4574-4578Abstract Full Text PDF PubMed Google Scholar), bind a Fe(II) chelate complex (25Aguirre J. Rodrı́guez R. Hansberg W. J. Bacteriol. 1989; 171: 6243-6250Crossref PubMed Google Scholar), or have a noncatalytic iron-sulfur cluster (27Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar). These modifications inactivate the enzymes and make them more susceptible to endogenous proteolytic activity (27Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar, 28Rivett A.J. J. Biol. Chem. 1987; 260: 300-305Abstract Full Text PDF Google Scholar, 29Davies K.J.A. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 30Roseman J.E. Levine R.L. J. Biol. Chem. 1985; 262: 2101-2110Abstract Full Text PDF Google Scholar). Safe disposal of H2O2 in cells is carried out by catalases and peroxidases. Hydrogen peroxide is formed mainly from dismutation of superoxide, which is generated by O2capturing an electron, usually from electron transport chains. Hydrogen peroxide is also a product of some oxidases. Being uncharged and not very reactive, it can diffuse between cell compartments. Its toxicity is traced to the formation of the hydroxyl radical upon capture of an electron, for instance, from Fe(II) or Cu(I). 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 (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). Thus, it is understandable why catalases are one of the most efficient enzymes known (31Fita I. Rossmann M.G. J. Mol. Biol. 1985; 185: 21-37Crossref PubMed Scopus (355) Google Scholar). It is so efficient that it cannot be saturated by H2O2at any concentration. Catalases are prevalent in most organisms (4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon Press, Oxford1989Google Scholar). Many microorganisms have more than one catalase, and in some a catalase is related to cell differentiation (32Loewen P.C. Triggs B, L. J. Bacteriol. 1984; 160: 668-675Crossref PubMed Google Scholar, 33Mulvey M.R. Switala J. Borys A. Loewen P.C. J. Bacteriol. 1990; 172: 6713-6720Crossref PubMed Google Scholar, 34von Ossowski I. Mulvey M.R. Leco P.A. Borys A. Loewen P.C. J. Bacteriol. 1991; 173: 514-520Crossref PubMed Scopus (126) Google Scholar, 35Bol D.K. Yasbin R.E. Gene (Amst.). 1991; 109: 31-37Crossref PubMed Scopus (39) Google Scholar, 36Engelmann S. Lindner C. Hecker M. J. Bacteriol. 1995; 177: 5598-5605Crossref PubMed Google Scholar, 37Hartig A. Ruis H. Eur. J. Biochem. 1986; 160: 487-490Crossref PubMed Scopus (73) Google Scholar, 38Cohen G. Rapatz W. Ruis H. Eur. J. Biochem. 1988; 176: 159-163Crossref PubMed Scopus (85) Google Scholar, 39Navarro R.E. Stringer M.A. Hansberg W. Timberlake W.E. Aguirre J. Curr. Genet. 1996; 29: 352-359PubMed Google Scholar, 40Kawasaki L. Wysong D. Diamond R. Aguirre J. J. Bacteriol. 1997; 179: 3284-3292Crossref PubMed Google Scholar, 41Fowler T. Rey M.W. Vaha-Vahe P. Power S.D. Berka R.M. Mol. Microbiol. 1993; 9: 989-998Crossref PubMed Scopus (48) Google Scholar, 42Redinbaugh M.G. Wadsworth G.J. Scandalius J.G. Biochim. Biophys. Acta. 1988; 951: 104-116Crossref PubMed Scopus (101) Google Scholar, 43Willekens H. Langebartels C. Tiré C. van Montagu M. Inzé D. van Camp W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10450-10454Crossref PubMed Scopus (171) Google Scholar, 44Loewen P.C. Switala J. Biochem. Cell Biol. 1988; 66: 707-714Crossref PubMed Scopus (23) Google Scholar, 45Hengge-Aronis R. Cell. 1993; 72: 165-168Abstract Full Text PDF PubMed Scopus (454) Google Scholar). Two catalase genes have been cloned in Aspergillus nidulans, one expressed only for asexual spores (conidia) (39Navarro R.E. Stringer M.A. Hansberg W. Timberlake W.E. Aguirre J. Curr. Genet. 1996; 29: 352-359PubMed Google Scholar, 40Kawasaki L. Wysong D. Diamond R. Aguirre J. J. Bacteriol. 1997; 179: 3284-3292Crossref PubMed Google Scholar). A third catalase activity has been recently found. 2L. Kawasaki and J. Aguirre, personal communication. 2L. Kawasaki and J. Aguirre, personal communication. 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We are indebted to Dr. Mario Rivera, Oklahoma State University, for help in the interpretation of the heme spectra.
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