Transcript Slide 1

Anaerobic Microbes:
Oxygen Detoxification Without
Superoxide Dismutase
Presented by
J. Spencer King
and
Seth I. Berger
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?
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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
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
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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
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Phylogenetic location
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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
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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
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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…
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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 desulfoferrodoxin’s 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
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 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.
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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
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
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 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].
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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 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
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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 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.
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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)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
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