Superoxide Inhibits 4Fe-4S Cluster Enzymes Involved in Amino Acid Biosynthesis

Abstract

Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1⌬ yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1⌬ mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPRdetectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.The antioxidant enzyme copper/zinc-superoxide dismutase 1 plays an integral role in the protection of many organisms from the oxidative aerobic environment.CuZn-SOD is localized to the cytosol, nucleus, and the intermembrane space (IMS) of the mitochondria, suggesting that it exerts its protective effect in multiple compartments (1). Another SOD that contains manganese (Mn-SOD or Sod2p) is located in the matrix of mitochondria. Saccharomyces cerevisiae lacking CuZn-SOD (sod1⌬) have distinct and well established aerobic phenotypes including diminished growth, auxotrophies for lysine and either methionine or cysteine, decreased ability to grow on nonfermentable carbon sources (2, 3), increased "free" (EPR-detectable) iron (4), hypersensitivity to millimolar concentrations of zinc (5), and exquisite sensitivity to redox-cycling drugs such as the herbicide paraquat (6, 7). All of these phenotypes (except zinc sensitivity) are also observed in mutants lacking the copper chaperone for CuZn-SOD 2 (lys7⌬ or ccs1⌬) and thus contain a form of the Sod1p polypeptide that is inactive because of a lack of copper in the active site (5, 8). Mutants lacking Sod2p have a less dramatic phenotype; they are sensitive to redox cycling drugs and grow poorly on nonfermentable carbon sources but show no aerobic auxotrophies (2).Many cellular components are susceptible to oxidative damage, including proteins, lipids, and DNA (9, 10). However, targets of superoxide-specific damage are much more limited. A particular type of protein prosthetic group, solvent-exposed 4Fe-4S clusters occurring in nonelectron transfer proteins, have been shown to be specifically damaged by superoxid...

Substantial DNA damage from submicromolar intracellular hydrogen peroxide detected in Hpx - mutants of Escherichia coli

“…Several of their growth defects were traced to the fact that superoxide rapidly destroys the [4Fe-4S] clusters of dehydratases (4)(5)(6)(7)(8). Similar injuries were subsequently discovered to occur during superoxide stress in higher organisms (9,10).…”

Copper Chaperone-Dependent and -Independent Activation of Three Copper-Zinc Superoxide Dismutase Homologs Localized in Different Cellular Compartments in Arabidopsis

“…Because of the limited sensitivity of the in-gel SOD activity assay, we used a more sensitive method, the Lys-independent aerobic growth assay, to detect the relatively weak CSD activity in yeast in vivo (Wallace et al, 2004). With this assay, Lys biosynthesis ability was maintained when ySOD1 activity was just higher than 2% of the wild-type level to rescue the Lys auxotrophy of sod1D (Corson et al, 1998).…”

“…Then, cells were centrifuged, washed, and resuspended in sterile water, and the A 600 was measured. For plate assays, the yeast cultures were diluted serially from optical density at 600 nm = 1 to 10 24 , plated on synthetic dropout Lys medium, and incubated at 30°C under aerobic conditions for 3 d. For the liquid assay, the yeast cultures were seeded from optical density at 600 nm = 0.01 in the Lys-lacking medium, incubated at 30°C for 24 h under aerobic conditions, and then cell density was determined by measuring the A 600 (Corson et al, 1998;Wallace et al, 2004).…”

The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival

“…Lysine auxotrophy in the sod1D single mutant of Sac. cerevisiae has been proposed to be caused by the inactivation of the 4Fe-4S cluster contained in the homoaconitase (encoded by the LYS4 gene) by O 2 $2 (Wallace et al, 2004). However, in Sch.…”

“…cerevisiae increases superoxide damage and that the homoaconitase Lys4, the second enzyme of the AAA pathway, is a superoxide-labile target (Wallace et al, 2004). We therefore decided to determine whether CgLys4 (CAGL0K10978g) was the cause of lysine auxotrophy in the sod1D sod2D mutant.…”