Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active site. Alternative mechanisms for the nitric oxide reductase (NOR) activity have been proposed consisting of either protonation of a diiron-bridging hyponitrite or “super-reduction” of a diferrous-dinitrosyl by the proximal FMNH2 in the rate-determining step. In order to test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron site but does not show multi-turnover NOR or O2 reductase (O2R) activity. Reactions of the reduced (diferrous) deflavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to 1 NO:diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = 1/2 EPR signal arising from antiferromagnetic coupling of an S = 3/2 {FeNO}7 with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N2O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O2R activities upon simple addition of FMN. The production of N2O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo-FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron-mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs.
An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H 2 O 2 and O 2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H 2 O 2 oxidoreductase) and NADH oxidase (NADH : O 2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H 2 O 2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H 2 O 2 and O 2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.
In this hemorrhage control education study, it was found that a short educational intervention can improve laypersons' self-efficacy and reported willingness to use a tourniquet in an emergency. Identified barriers to act should be addressed when designing future hemorrhage control public health education campaigns. Community education should continue to be a priority of the "Stop the Bleed" campaign. Ross EM , Redman TT , Mapp JG , Brown DJ , Tanaka K , Cooley CW , Kharod CU , Wampler DA . Stop the bleed: the effect of hemorrhage control education on laypersons' willingness to respond during a traumatic medical emergency. Prehosp Disaster Med. 2018;33(2):127-132.
Acrylamide, a neurotoxin and suspected carcinogen, is produced by industrial processes and during the heating of foods. In this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions (doubling time of 10 to 12 h) but poorly under anaerobic dark or aerobic conditions. Acrylamide was rapidly deamidated to acrylate by strain Ac1, and the subsequent degradation of acrylate was the rate-limiting reaction in cell growth. Acrylamide metabolism by succinate-grown cultures occurred only after a lag period, and the induction of acrylamide-degrading activity was prevented by the presence of protein or RNA synthesis inhibitors. 13 Acrylamide is a toxic three-carbon compound containing an amide group and an ␣,-unsaturated olefin bond. It exerts its toxic effects by forming adducts to nucleophilic moieties such as sulfhydryl groups on proteins (4). Acrylamide is potent neurotoxin (30) and suspected to be a carcinogen (21, 27). Although acrylamide is not typically found in nature, it is widely used in industrial processes as a polymerizing agent, hardener, and as a flocculent in water treatment. Accordingly, acrylamide released from industrial processes has resulted in the contamination of both soils and aquatic environments (6). Recently, it was discovered that acrylamide is formed in starchy foods after being cooked at the high temperatures required for frying and baking (26,27). These foods, which have undetectable levels before cooking, have acrylamide levels as high as 2.3 mg/kg after cooking (25). The formation of acrylamide is believed to be a product of the reaction between asparagine and glucose during the cooking process (14, 24).In spite of acrylamide's toxicity in the monomer form, some microorganisms are able to utilize acrylamide as their sole carbon source for growth (16,17,22,32,33). All studies conducted to date show that there is an initial deamidation step that converts acrylamide to acrylic acid (acrylate) (15,17,22,33). The subsequent fate of acrylate has not been well defined for acrylamideutilizing bacteria but probably involves pathways and enzymes that have been characterized to various degrees for other bacteria capable of acrylate catabolism. In aerobic acrylate-utilizing bacteria, acrylate metabolism has been shown to proceed via hydroxylation to -hydroxypropionate, which is further oxidized to CO 2 (1, 2). Highlighting a different fate for acrylate under anaerobic conditions, Clostridium propionicum is capable of fermenting acrylate to acetate and propionate (10). Acrylate has also been shown to serve as a terminal electron acceptor for Desulfovibrio acrylicus, producing propionate which is not further metabolized as a growth substrate (31). Cell extracts of C. propionicum have been shown to dehydrate (R)-lactate to acrylate (20)...
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