Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

Abstract

Methanogenic Archaea are ancient microorganisms that retain many features of their ancestors and exist under extreme conditions of severe anaerobiosis. However, in spite of their inability to produce CH 4 in the presence of oxygen, they are capable of existing in the presence of oxygen for a short time. Methanobrevibacter arboriphilus and Methanobacterium thermoautotrophicum were shown to maintain their vitality under aerobic con ditions for several hours [1]. In the case of Methanosarcina barkeri, this period was more than 24 h, this being due to the formation of cell clots [1]. To provide defense against the toxic effects of prod ucts of incomplete reduction of oxygen (H 2 O 2 , OH • , and O 2 ), many strict anaerobes as well as aerobic organisms contain antioxidant enzymes. The most important of these are catalase and superoxide dismutase (SOD). SOD removes free superoxide radicals that arise during the one electron reduction of O 2 , catalyzing the dismu tation reaction of O 2 , yielding H 2 O 2 and O 2 . Hydrogen peroxide acts as a reductant of the enzyme at low con centrations and as a reversible inactivator at high con centrations. The reaction mechanism includes alternat ing reduction and reoxidation of the metal in the active site of the SOD during consecutive interactions with O 2 [8].The detection of SOD in obligate anaerobes raised a question concerning its function and origin in these organisms. Apparently, the main physiological role of SOD in anaerobic microorganisms as well as in aerobes is the prevention of cell damage caused by oxygen by neu tralizing superoxide radicals. The latter may arise due to a transient exposure of the anaerobic cells to the air during the reaction of O 2 with reduced flavins, catecholamines, quinones, iron and sulfur containing proteins, as well as under the action of mutagens and UV radiation [9]. It has been shown that strictly anaerobic microorganisms pos sessing significant SOD activity exhibit moderate or high ISSN 0006 2979, Biochemistry (Moscow), 2006, Vol. 71, No. 4, pp. 441 447. © Pleiades Publishing, Inc., 2006. Original Russian Text © A. L. Brioukhanov, V. J. Nesatyy, A. I. Netrusov, 2006, published in Biokhimiya, 2006 441 * To whom correspondence should be addressed. Abstract-Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inac tivates the enzyme, which is consistent with the properties of the known Fe containing SODs from methanogenic Archaea. Purification and Characterization of Fe Containing

The catalase and superoxide dismutase genes are transcriptionally up-regulated upon oxidative stress in the strictly anaerobic archaeon Methanosarcina barkeri

“…We have shown here that M. barkeri can grow after addition of 0?8 mM H 2 O 2 or of 100 mM PQ to the culture medium. M. barkeri has a SOD activity which is two times lower than the specific SOD activity in Methanobrevibacter arboriphilus (Brioukhanov & Netrusov, 2004), and M. barkeri is nearly five times more sensitive to PQ exposure compared to M. arboriphilus (Brioukhanov et al, 2006). This confirms the relationship between SOD activity in the cells and aerotolerance of the micro-organism.…”

“…Generally, microbial species with high SOD activity have high or moderate aerotolerance in comparison to species with low or no SOD activity (Hewitt & Morris, 1975;Privalle & Gregory, 1979;Gregory & Dapper, 1980). Fe-containing SODs have been purified and characterized in the following methanogenic archaea: Methanobacterium bryantii (Kirby et al, 1981), Methanobacterium thermoautotrophicum (Takao et al, 1991), Methanosarcina barkeri (Brioukhanov et al, 2000) and Methanobrevibacter arboriphilus (Brioukhanov et al, 2006). Catalase (EC 1.11.1.6) catalyses the conversion of 2 H 2 O 2 to O 2 and H 2 O, using H 2 O 2 as the electron donor.…”

Air-Adapted Methanosarcina acetivorans Shows High Methane Production and Develops Resistance against Oxygen Stress

“…Basal activities of SOD and CAT as well as increased activities induced by oxidant stressors have been also found in Methanosarcina barkeri [ 13 ] and other methanogens [ 12 , 37 ]. M .…”

“…In gel enzyme activities indicated that Zn 2+ and Fe 2+ increase SOD activity, as reported for the M . arboriphilus enzyme [ 37 ]. There are genes annotated for Zn/Cu–dependent SOD (MA2422) and Fe/Mn-dependent SOD (MA1574) in the genome of M .…”

Evolutive and structural characterization of Nostoc commune iron-superoxide dismutase that is fit for modification

“…Three classes of SODs occur (Fe-SOD, Mn-SOD, and Cu/ Zn-SOD), each differing in the metals at their catalytic active sites. Fe-SOD and Mn-SOD are found in Eubacteria, Eubacteria-derived eukaryotic mitochondria, and Archaebacteria (Regelsberger et al, 2004;Gottlieb et al, 2005;Brioukhanov et al, 2006;Chen et al, 2007;Seo et al, 2007). The Cu/Zn enzyme is confined almost exclusively to the cytoplasm of eukaryotic organisms.…”