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Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site.

Rühl, Patrick and Haas, Patrick and Seipel, Dominik and Becker, Jan and Kletzin, Arnulf (2018):
Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site.
In: Frontiers in microbiology, p. 1610, 9, ISSN 1664-302X,
[Article]

Abstract

Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for HS detoxification in mitochondria. One of the two -genes of the acidophilic bacterium was expressed in The protein (PDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein () at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. for GSH and were 0.5 mM and 181 s, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a of 122.3 U/mg protein, a of 198 s and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the RA, DA, and HA variants were inactive concomitant to a decrease of their melting points by 3-8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C and C). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides -ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C, C, and C by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the CA and CA variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

Item Type: Article
Erschienen: 2018
Creators: Rühl, Patrick and Haas, Patrick and Seipel, Dominik and Becker, Jan and Kletzin, Arnulf
Title: Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site.
Language: English
Abstract:

Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for HS detoxification in mitochondria. One of the two -genes of the acidophilic bacterium was expressed in The protein (PDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein () at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. for GSH and were 0.5 mM and 181 s, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a of 122.3 U/mg protein, a of 198 s and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the RA, DA, and HA variants were inactive concomitant to a decrease of their melting points by 3-8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C and C). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides -ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C, C, and C by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the CA and CA variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

Journal or Publication Title: Frontiers in microbiology
Volume: 9
Divisions: 10 Department of Biology
10 Department of Biology > Sulfur Biochemistry and Microbial Bioenergetics
Date Deposited: 28 Aug 2018 06:25
Identification Number: pmid:30072973
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