Anaerobic Microbes: Oxygen Detoxification Without Superoxide Dismutase

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Anaerobic Microbes Oxygen Detoxification
Without Superoxide Dismutase

• Presented by
• J. Spencer King
• and
• Seth I. Berger

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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
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Verbosity to obscure ignorance

• will not be tolerated.

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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
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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.

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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
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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

Berger-King 9.17.03
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Phylogenetic location
Berger-King 9.17.03
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Superoxide O2-

• Present in all aerobic environments
• Molecular oxygen has strong reduction 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
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Superoxide Dismutase and Catalase

• Aerobic organism defense superoxide removal
  enzyme.
• SOD removes O2-
• Catalase then processes the H2O2 product
• In some instances, non-specific peroxidases
  process the H2O2

Berger-King 9.17.03
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SOD and Catalase in Anaerobes

• SOD and catalase genes not present in completed
  anaerobic genomes circa 1999
• Why?

Both produce Oxygen!

• Strict Anaerobes need some other method of
  removing toxic oxygen species

Berger-King 9.17.03
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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)

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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)

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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 purified iron-containing redox protein

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Sequence Homologies

• 40 identity to desulfoferrodoxins iron
  containing COOH-terminal region
• 50 identity to neelaredoxin
• Both are redox proteins and have been shown to
  posses SOD activity.

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Detecting SOD Activity

• Standard SOD Assay
• Steady-state generation of superoxide
• 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

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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

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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
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Comparison of Different Assay Results
Superoxide source Superoxide detection method Specific Activity Specific Activity
Superoxide source Superoxide detection method Bovine SOD P. Furiousus SOR
Xanthine oxidase Cytochrome c reduction 3400 4000
Pyrogallol 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
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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)

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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 A490
• Also autooxidizes in air
• SOR increased rate of oxidation
• Effect of SOR required superoxide
• SOD decreased rate

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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 O2 producing catalase

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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
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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.
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Superoxide Reductase

• SOR and NROR are both catalytically active and
  efficient at 25 C.
• 75 C cooler than P. furiosus growth
  temperature.
• Exposure to O2 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.

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Critiques

• Sequence comparisons
• -similarity is not shown.
• Sequence analysis methods not detailed
• What to do with the H2O2 ?
• Only hypothetical peroxidases
• Peroxidase activity at 25C?
• Formatting and layout
• Diagrams are informative but not attractive
• More detailed materials and methods
• Science publication requirements.
• Fortuitousness of Fig 1 line B,3

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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
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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)Hrubredoxin 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
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