Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K⁺ channel responsible for K⁺ efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca²⁺, tetraethylammonium, Ba²⁺, Cs⁺ and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K⁺ channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K⁺ efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K⁺-channel activation. In animals, activation of K⁺-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K⁺-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K⁺ channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K⁺ channels.

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of ⁵⁵Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe²⁺-Fur complex.

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap α-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2′-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F₂-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F₂-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F₂-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F₂-isoprostanes by ∼80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it’s concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.