Anaerobic Microbes- Oxygen Detoxification Without .ppt
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1、Anaerobic Microbes: Oxygen Detoxification Without Superoxide Dismutase,Presented by J. Spencer King and Seth I. Berger,Before we begin a few questions,Why dont pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides?SOR in p. furiosus functions efficiently 75 C below the optima
2、l growth temperature of p. furiosus. Why do the authors of the paper believe this is so?,Berger-King 9.17.03,Verbosity to obscure ignorance,will not be tolerated.,Before we begin a few questions,Why dont pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides?SOR in p. furiosus
3、 functions efficiently 75 C below the optimal growth temperature of p. furiosus. Why do the authors of the paper believe this is so?,Berger-King 9.17.03,Answers,Because SOD and Catalase both produce Oxygen.The only time that p. furiosus is exposed to oxygen is when the deep sea vent waters mix with
4、the surrounding cold seawater.,Brief Synopsis of Anaerobes,Aerotolerant Anaerobes O2 not Toxic O2 independent metabolism Facultative Anaerobes Can grow with or without O2 Change metabolism depending on O2 concentration Strict Anaerobes O2 is Toxic,Berger-King 9.17.03,Berger-King 9.17.03,About Pyroco
5、ccus furiosus,Archea Strict Anaerobe Hyperthermophilic Deep sea vents 70 to 100 C Up to 200 atm Irregular cocci shape Polar flagella group Hydrogen important in metabolism,Phylogenetic location,Berger-King 9.17.03,Superoxide O2-,Present in all aerobic environments Molecular oxygen has strong reducti
6、on activity Unstable free radical very toxic Reacts with H2O2 to from hydroxyl radicals Anaerobic organisms need protection too Exposure to oxygen sometime during life cycle is possible especially for microbes living in water, like Pyrococcus furiosus,Berger-King 9.17.03,Superoxide Dismutase and Cat
7、alase,Aerobic organism defense superoxide removal enzyme. SOD removes O2- Catalase then processes the H2O2 productIn some instances, non-specific peroxidases process the H2O2,Berger-King 9.17.03,SOD and catalase genes not present in completed anaerobic genomes circa 1999 Why?,SOD and Catalase in Ana
8、erobes,Berger-King 9.17.03,Strict Anaerobes need some other method of removing toxic oxygen species,Both produce Oxygen!,Requirements for SOD replacement,Remove superoxide before it becomes toxic Do not produce oxygen Be active under the conditions required by Pyrococcus furiosusData suggests the me
9、chanism for oxygen metabolism in Pyrococcus furiosus is Superoxide Reductase (SOR),Preliminary Steps,Select model organism P. furiosis: a strictly anaerobic hyperthermophile Isolate Putative Superoxide Dismutase(SOD) Multistep Column Chromatography Denaturing Gel Electrophoresis 14,000 Daltons Direc
10、t Chemical Analysis Contains Iron ( 0.5 atoms/mol) found using a inductively coupled argon plasma spectrometer (ICAP),Preliminary Steps,Clone gene NH2-terminal amino acid sequence information Locate in known genome 124 amino acid protein(14,323 Da) 14 bp downstream of rubredoxin (5895 Da) Previously
11、 purified iron-containing redox protein,Sequence Homologies,40% identity to desulfoferrodoxins iron containing COOH-terminal region 50% identity to neelaredoxinBoth are redox proteins and have been shown to posses SOD activity.,Detecting SOD Activity,Standard SOD Assay Steady-state generation of sup
12、eroxide Bovine Xanthine Oxidase + Xanthine Superoxide reduces Cytochrome C directly Measure A550 increase rate One unit of Activity is amount of protein needed to inhibit rate by 50%,Differences Between SOD and SOR,SOR does not oxidize Cytochrome C when it was initially reduced with Sodium Dithionit
13、e. It will subsequently oxidize it when a superoxide source is added. No Oxygen is generated Different behaviors in Assays,Bovine SOD vs P. furiosus SOR,Figure 1. Pyrococcus furiosus superoxide reductase is not a superoxide dismutase. Reactions were performed as described (18) in 1-ml cuvettes under
14、 aerobic conditions. Superoxide produced by xanthine (0.2 mM) and xanthine oxidase (3.4 g) directly reduced horse heart cytochrome c (20 M), as shown by the increase in absorbance at 550 nm (A550) (A and B, trace 1). Addition of bovine SOD (3.4 g, 1 U) inhibited the rate of reduction (A), trace 2. E
15、xcess SOD (40 U) prevented reduction completely (A), trace 3, and additional SOD (60 U) had no further effect (A), trace 4. P. furiosus SOR (2.5 g or 17 nM) also resulted in inhibition of reduction (B), trace 2, and more SOR (6.2 g) completely prevented reduction (B), trace 3. Addition of excess SOR
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