Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.
Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (<5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70°C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.A diverse set of bacteria possess the ability to oxidize CO to CO2 (for reviews, see references 10 through 12, 14, 15, 18, 26, and 30). In acetogenic and methanogenic bacteria, CO is oxidized by multisubunit nickel-containing CO dehydrogenase complexes (8,9,21). The expression of these enzymes is not affected by the presence or absence of CO, and these enzymes appear to be involved in acetate metabolism, with CO oxidation being a secondary reaction (15,29,30).Aerobic carboxydotrophic bacteria oxidize CO by using carbon monoxide oxidase, an oxygen-stable iron-sulfur enzyme containing flavin and molybdopterin (16)(17)(18). In these bacteria CO oxidase is induced by the presence of CO, and hydrogenase is induced in cells grown with CO or with CO2 and H2 (18).Some photosynthetic bacteria tolerate CO (13), and Rhodocyclus (formerly Rhodopseudomonas) gelatinosus has been shown by Uffen and co-workers to utilize CO as its sole carbon and energy source during anaerobic growth in the dark (25). The membrane-bound CO-oxidizing system of R. gelatinosus is inducible by CO and produces CO2 and H2 as products of the oxidation of CO (27,28). CO-dependent evolution of H2 from extracts of Rhodospirillum rubrum S1 has been noted (26). We have previously reported COdependent formation of H2 and CO2 by R. rubrum cells grown in the light on ammonium-malate medium (6) and that exposure of light-grown R. rubrum cultures to CO led to significantly increased levels of CO dehydrogenase (4).In this paper these initial observations are extended to demonstrate that CO induces two enzymatic activities, CO dehydrogenase and a CO-insensitive hydrogenase, which appear to function together to accomplish the oxidation of CO to CO2 and H2. These activities are inactivated by 02 both in vivo and in vitro, and the synthesis of CO dehydrogenase is shown to be inhibited by oxygen. Chromatophore suspensions. Membrane preparations (chromatophores) were derived from cells which had been treated with CO for 24 h before harvest. Cells were broken and the chromatophores were collected by centrifugation as previously described (4) and stored in liquid nitrogen.Enzyme assays. CO dehydrogenase activity was assayed by using a CO-dependent methyl viologen reduction assay as previously described (6). Cells were lysed by grinding before the assay for CO dehydrogenas...
A second nitrogenase activity has been demonstrated in Rhodospirillum rubrum. This nitrogenase is expressed whenever a strain lacks an active Mo nitrogenase because of physiological or genetic inactivation. The alternative nitrogenase is able to support growth on N2 in the absence of fixed N. V does not stimulate, nor does Mo or W inhibit, growth or activity under the conditions tested. The proteins responsible for this activity were identified by electrophoretic and immunological properties. The synthesis of these proteins was repressed by NH4'. The alternative nitrogenase reductase is ADP ribosylated in response to darkness by the system that regulates the activity of the Mo nitrogenase. The genes for the alternative nitrogenase have been cloned, and the alternative nitrogenase reductase has been expressed in an in vitro transcription-translation system.
The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent ␣ 2  2 ␥ 2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum-or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an ␣ 2 dimer containing an Fe 4 S 4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H 2 very well but reduces N 2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.
The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-'4C]acetate, hydrogen, and ATP formed '4CH4 and ['4C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95 % purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.
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