Anaerobic Microbes Oxygen Detoxification Without Superoxide Dismutase Presented

Anaerobic Microbes: Oxygen Detoxification Without Superoxide Dismutase Presented by J. Spencer King and Seth I. Berger-King 9. 17. 03

Before we begin… a few questions l Why don't pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides? l 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

Verbosity to obscure ignorance will not be tolerated. Berger-King 9. 17. 03

Before we begin… a few questions l Why don't pure anaerobes use SOD to remove superoxide, and Catalase to remove Peroxides? l 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

Answers l Because SOD and Catalase both produce Oxygen. l The only time that p. furiosus is exposed to oxygen is when the deep sea vent waters mix with the surrounding cold seawater. Berger-King 9. 17. 03

Brief Synopsis of Anaerobes l Aerotolerant Anaerobes ¡ O 2 not Toxic ¡ O 2 independent metabolism l Facultative Anaerobes ¡ Can grow with or without O 2 ¡ Change metabolism depending on O 2 concentration l Strict Anaerobes ¡ O 2 is Toxic Berger-King 9. 17. 03

About Pyrococcus furiosus Archea l Strict Anaerobe l Hyperthermophilic l ¡ Deep sea vents l l 70° to 100° C Up to 200 atm Irregular cocci shape l Polar flagella group l Hydrogen important in metabolism l Berger-King 9. 17. 03

Phylogenetic location Berger-King 9. 17. 03

Superoxide O 2 - l Present in all aerobic environments ¡ Molecular oxygen has strong reduction activity l Unstable free radical – very toxic ¡ Reacts with H 2 O 2 to from hydroxyl radicals l 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 Catalase Aerobic organism defense superoxide removal enzyme. l SOD removes O 2 - l l Catalase then processes the H 2 O 2 product l In some instances, non-specific peroxidases process the H 2 O 2 Berger-King 9. 17. 03

SOD and Catalase in Anaerobes l SOD and catalase genes not present in completed anaerobic genomes circa 1999 l Why? Both produce Oxygen! l Strict Anaerobes need some other method of removing toxic oxygen species… Berger-King 9. 17. 03

Requirements for SOD replacement Remove superoxide before it becomes toxic l Do not produce oxygen l Be active under the conditions required by Pyrococcus furiosus l l Data suggests the mechanism for oxygen metabolism in Pyrococcus furiosus is Superoxide Reductase (SOR) Berger-King 9. 17. 03

Preliminary Steps l Select model organism ¡ l P. furiosis: a strictly anaerobic hyperthermophile Isolate Putative Superoxide Dismutase(SOD) ¡ ¡ ¡ Multistep Column Chromatography Denaturing Gel Electrophoresis l ~14, 000 Daltons Direct Chemical Analysis l Contains Iron ( 0. 5 atoms/mol) found using a inductively coupled argon plasma spectrometer (ICAP) Berger-King 9. 17. 03

Preliminary Steps l Clone gene ¡ NH 2 -terminal amino acid sequence information ¡ Locate in known genome 124 amino acid protein(14, 323 Da) l 14 bp downstream of rubredoxin (5895 Da) l Previously purified iron-containing redox protein Berger-King 9. 17. 03

Sequence Homologies l 40% identity to desulfoferrodoxin’s iron containing COOH-terminal region l 50% identity to neelaredoxin Both are redox proteins and have been shown to posses SOD activity. Berger-King 9. 17. 03

Detecting SOD Activity l Standard SOD Assay ¡ Steady-state generation of superoxide l 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% Berger-King 9. 17. 03

Differences Between SOD and SOR l 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. l No Oxygen is generated l Different behaviors in Assays Berger-King 9. 17. 03

Bovine SOD vs P. furiosus SOR SOD behavior SOR behavior 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 m. M) and xanthine oxidase (3. 4 µg) directly reduced horse heart cytochrome c (20 µM), as shown by the increase in absorbance at 550 nm (A 550) (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 n. M) 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, A 550 = 0. 178 for fully oxidized cytochrome c. Berger-King 9. 17. 03

Comparison of Different Assay Results Superoxide source Superoxide detection method Xanthine oxidase Specific Activity Bovine SOD P. Furiousus SOR Cytochrome c reduction 3400 4000 Pyrogallol oxidation 2300 80 Xanthine oxidase Epinephrine oxidation 2200 100 Xanthine oxidase Nitroblue tetrazolium reduction 1800 200 Xanthine oxidase Acetylated Cytochrome c reduction 3400 100 Berger-King 9. 17. 03

Other Genomes l Homologues are found in almost all complete genomes from anaerobes and a couple incomplete ones. ¡ 116 – 138 Residues with 20 – 70% identity l Not found in any of the 16 available genomes of true aerobes (circa 1999) Berger-King 9. 17. 03

Rubredoxin l Adjacent to SOR in P. Furiosus genome l Known Electron Carrier l Oxidized by Superoxide ¡ (opposed to cytochrome C which is reduced) ¡ Can be measured by A 490 l Also autooxidizes in air l SOR increased rate of oxidation ¡ Effect of SOR required superoxide ¡ SOD decreased rate Berger-King 9. 17. 03

Rubredoxin Found in almost ever known anaerobic genome despite function previously unknown. l NADP-ruberedoxin oxioreductase reduced rubredoxin. l ¡ l 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 Berger-King 9. 17. 03

Bovine SOD vs P. furiosus SOR SOD behavior SOR behavior Figure 2. Pyrococcus furiosus SOR is a rubredoxinsuperoxide 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 A 490. 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]. 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; XH 2, unknown organic electron donor. Enzymes and proteins shown in bold were purified from P. furiosus; the others are hypothetical, based on genome sequence analyses. Berger-King 9. 17. 03

Superoxide Reductase l SOR and NROR are both catalytically active and efficient at 25° C. ¡ ~75° C cooler than P. furiosus growth temperature. l 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. Berger-King 9. 17. 03

Critiques l Sequence comparisons ¡ ¡ l What to do with the H 2 O 2 ? ¡ l Only hypothetical peroxidases l Peroxidase activity at 25°C? Formatting and layout ¡ ¡ l %-similarity is not shown. Sequence analysis methods not detailed Diagrams are informative but not attractive More detailed materials and methods l Science publication requirements. Fortuitousness of Fig 1 line B, 3 Berger-King 9. 17. 03

Bovine SOD vs P. furiosus SOR SOD behavior SOR behavior 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 m. M) and xanthine oxidase (3. 4 µg) directly reduced horse heart cytochrome c (20 µM), as shown by the increase in absorbance at 550 nm (A 550) (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 n. M) 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, A 550 = 0. 178 for fully oxidized cytochrome c. Berger-King 9. 17. 03

Follow up Article 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 Berger-King 9. 17. 03

Berger-King 9. 17. 03