1. Berger-King 9.17.03 Anaerobic Microbes: Oxygen Detoxification Without Superoxide Dismutase Presented by J. Spencer King and Seth I. Berger

2. Berger-King 9.17.03 Before we begin… a few questions Why don't pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides? SOR in p. furiosus 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

3. Verbosity to obscure ignorance will not be tolerated.

4. Berger-King 9.17.03 Before we begin… a few questions Why don't pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides? SOR in p. furiosus 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

5. 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 the surrounding cold seawater.

6. Berger-King 9.17.03 Brief Synopsis of Anaerobes Aerotolerant Anaerobes  O 2 not Toxic  O 2 independent metabolism Facultative Anaerobes  Can grow with or without O 2  Change metabolism depending on O 2 concentration Strict Anaerobes  O 2 is Toxic Berger-King 9.17.03

7. About Pyrococcus 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

8. Berger-King 9.17.03 Phylogenetic location Berger-King 9.17.03

9. Superoxide O 2 - Present in all aerobic environments  Molecular oxygen has strong reduction activity Unstable free radical – very toxic  Reacts with H 2 O 2 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

10. Superoxide Dismutase and Catalase Aerobic organism defense superoxide removal enzyme. SOD removes O 2 - Catalase then processes the H 2 O 2 product In some instances, non-specific peroxidases process the H 2 O 2 Berger-King 9.17.03

11. SOD and catalase genes not present in completed anaerobic genomes circa 1999 Why? SOD and Catalase in Anaerobes Berger-King 9.17.03 Strict Anaerobes need some other method of removing toxic oxygen species… Both produce Oxygen!

12. Berger-King 9.17.03 Requirements for SOD replacement Remove superoxide before it becomes toxic Do not produce oxygen Be active under the conditions required by Pyrococcus furiosus Data suggests the mechanism for oxygen metabolism in Pyrococcus furiosus is Superoxide Reductase (SOR)

13. Berger-King 9.17.03 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  Direct Chemical Analysis Contains Iron ( 0.5 atoms/mol) found using a inductively coupled argon plasma spectrometer (ICAP)

14. Berger-King 9.17.03 Preliminary Steps Clone gene  NH 2 -terminal amino acid sequence information  Locate in known genome 124 amino acid protein(14,323 Da) 14 bp downstream of rubredoxin (5895 Da)  Previously purified iron-containing redox protein

15. Berger-King 9.17.03 Sequence Homologies 40% identity to desulfoferrodoxin’s iron containing COOH-terminal region 50% identity to neelaredoxin Both are redox proteins and have been shown to posses SOD activity.

16. Berger-King 9.17.03 Detecting SOD Activity Standard SOD Assay  Steady-state generation of superoxide Bovine Xanthine Oxidase + Xanthine  Superoxide reduces Cytochrome C directly  Measure A 550 increase rate  One unit of Activity is amount of protein needed to inhibit rate by 50%

17. Berger-King 9.17.03 Differences Between SOD and SOR SOR does not oxidize Cytochrome C when it was initially reduced with Sodium Dithionite.  It will subsequently oxidize it when a superoxide source is added. No Oxygen is generated Different behaviors in Assays

18. Berger-King 9.17.03 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 aerobic conditions. Superoxide produced by xanthine (0.2 mM) and xanthine oxidase (3.4 µg) directly reduced horse heart cytochrome c (20 µM), as shown by the increase in absorbance at 550 nm (A550) (A and B, trace 1). Addition of bovine SOD (3.4 µg, 1 U) inhibited the rate of reduction [(A), trace 2]. Excess 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 µg or 17 nM) also resulted in inhibition of reduction [(B), trace 2], and more SOR (6.2 µg) completely prevented reduction [(B), trace 3]. Addition of excess SOR (15 µg) caused oxidation of the reduced cytochrome c that was present before SOR addition [(B), trace 4]. Time zero is when SOR or SOD was added to the cuvettes (approximately 90 s after addition of xanthine oxidase). Under these conditions, A550 = 0.178 for fully oxidized cytochrome c. SOD behavior SOR behavior

19. Berger-King 9.17.03 Comparison of Different Assay Results Superoxide source Superoxide detection method Specific Activity Bovine SODP. Furiousus SOR Xanthine oxidase Cytochrome c reduction 34004000 PyrogallolPyrogallol oxidation230080 Xanthine oxidase Epinephrine oxidation 2200100 Xanthine oxidase Nitroblue tetrazolium reduction 1800200 Xanthine oxidase Acetylated Cytochrome c reduction 3400100

20. Berger-King 9.17.03 Other Genomes Homologues are found in almost all complete genomes from anaerobes and a couple incomplete ones.  116 – 138 Residues with 20 – 70% identity Not found in any of the 16 available genomes of true aerobes (circa 1999)

21. Berger-King 9.17.03 Rubredoxin Adjacent to SOR in P. Furiosus genome Known Electron Carrier Oxidized by Superoxide  (opposed to cytochrome C which is reduced)  Can be measured by A 490 Also autooxidizes in air SOR increased rate of oxidation  Effect of SOR required superoxide  SOD decreased rate

22. Berger-King 9.17.03 Rubredoxin Found in almost ever known anaerobic genome despite function previously unknown. NADP-ruberedoxin oxioreductase reduced rubredoxin.  Provides a mechanism for providing the reducing power for superoxide reduction. HOWEVER, still produces peroxide  Must be removed, but not via O 2 producing catalase

23. Berger-King 9.17.03 Bovine SOD vs P. furiosus SOR Figure 2. Pyrococcus furiosus SOR is a rubredoxin- superoxide oxidoreductase. Reactions were done as in Fig. 1, except that reduced rubredoxin replaced cytochrome c. Superoxide directly oxidized P. furiosus rubredoxin, as shown by the increase in A490. Rubredoxin (28 µM) reduced by the addition of sodium dithionite (42 µM) slowly auto-oxidized upon exposure to air (A and B, trace 1). Addition of superoxide rapidly increased the rate of oxidation [(A) and (B), trace 2]. Catalase (10 U) had little effect [(A), trace 5], whereas in a separate experiment, bovine SOD (1 U) abolished the effect of superoxide [(A), trace 3], and excess SOD (10 U) slowed down even the spontaneous oxidation of rubredoxin [(A), trace 4]. In contrast, addition of P. furiosus SOR (1.2 µg) increased the rate of superoxide-dependent rubredoxin oxidation [(B), trace 3], and the rate increased with additional SOR [1.2 µg; (B), trace 4]. SOD behavior SOR behavior

24. Berger-King 9.17.03 Detoxification System Figure 3. Model for detoxification of reactive oxygen species in anaerobes such as P. furiosus. Abbreviations are as follows: NROR, NAD(P)H-rubredoxin oxidoreductase; Rdred, reduced rubredoxin; Rdox, oxidized rubredoxin; XH2, unknown organic electron donor. Enzymes and proteins shown in bold were purified from P. furiosus; the others are hypothetical, based on genome sequence analyses.

25. Berger-King 9.17.03 Superoxide Reductase SOR and NROR are both catalytically active and efficient at 25° C.  ~75° C cooler than P. furiosus growth temperature. Exposure to O 2 in the deep sea vents is limited to cold exposure to seawater  SOR and NROR together are a constitutively expressed defense mechanism which becomes active when the cell is exposed to a hostile environment.

26. Berger-King 9.17.03 Critiques Sequence comparisons  %-similarity is not shown.  Sequence analysis methods not detailed What to do with the H 2 O 2 ?  Only hypothetical peroxidases Peroxidase activity at 25°C? Formatting and layout  Diagrams are informative but not attractive  More detailed materials and methods Science publication requirements. Fortuitousness of Fig 1 line B,3

27. Berger-King 9.17.03 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 aerobic conditions. Superoxide produced by xanthine (0.2 mM) and xanthine oxidase (3.4 µg) directly reduced horse heart cytochrome c (20 µM), as shown by the increase in absorbance at 550 nm (A550) (A and B, trace 1). Addition of bovine SOD (3.4 µg, 1 U) inhibited the rate of reduction [(A), trace 2]. Excess 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 µg or 17 nM) also resulted in inhibition of reduction [(B), trace 2], and more SOR (6.2 µg) completely prevented reduction [(B), trace 3]. Addition of excess SOR (15 µg) caused oxidation of the reduced cytochrome c that was present before SOR addition [(B), trace 4]. Time zero is when SOR or SOD was added to the cuvettes (approximately 90 s after addition of xanthine oxidase). Under these conditions, A550 = 0.178 for fully oxidized cytochrome c. SOD behavior SOR behavior

28. Berger-King 9.17.03 June 2002: “The evidence for superoxide reduction by SOR is now overwhelming and comes from a variety of anaerobic and microaerophilic species...” “The catalytic Fe site of SOR is structurally and electronically tuned to mediate superoxide reduction rather than oxidation...” “NAD(P)H, via rubredoxin and NAD(P)H:rubredoxin oxidoreductase [is] the source of reductant...” “What is still to be determined is the fate of the peroxide generated by the SOR reaction…” Journal of Biological Inorganic Chemistry Issue: Volume 7, Number 6 Date: June 2002 Pages: 647 - 652 Follow up Article

29. Berger-King 9.17.03