Berger-King 9.17.03

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?

Berger-King 9.17.03

Berger-King 9.17.03

Verbosity to obscure ignorance

will not be tolerated.

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

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 genes not present in completed anaerobic genomes circa 1999

Why?

SOD and Catalase in Anaerobes

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

Both produce Oxygen!

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Requirements for SOD replacement

Remove superoxide before it becomes toxicDo not produce oxygenBe 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 generatedDifferent 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

Bovine SOD P. Furiousus SOR

Xanthine oxidaseCytochrome c

reduction3400 4000

Pyrogallol Pyrogallol oxidation 2300 80

Xanthine oxidaseEpinephrine

oxidation2200 100

Xanthine oxidaseNitroblue tetrazolium

reduction1800 200

Xanthine oxidaseAcetylated

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 genomeKnown Electron CarrierOxidized by Superoxide

(opposed to cytochrome C which is reduced) Can be measured by A490

Also autooxidizes in airSOR 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 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

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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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 ChemistryIssue: Volume 7, Number 6 Date: June 2002 Pages: 647 - 652

Follow up Article

Berger-King 9.17.03