Persulfide Dioxygenase from Acidithiobacillus Caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Acti

fmicb-09-01610 July 18, 2018 Time: 16:14 # 1 ORIGINAL RESEARCH published: 20 July 2018 doi: 10.3389/fmicb.2018.01610 Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site Patrick Rühl, Patrick Haas, Dominik Seipel, Jan Becker and Arnulf Kletzin* Department of Biology, Sulfur Biochemistry and Microbial Bioenergetics, Technische Universität Darmstadt, Darmstadt, Germany Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for H2S detoxification in mitochondria. One of the two pdo-genes of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg ◦ protein (Vmax) at 40 C and pH 7.5 with sulfur and GSH following Michaelis–Menten −1 Edited by: kinetics. KM for GSH and Kcat were 0.5 mM and 181 s , respectively. Glutathione Christiane Dahl, persulfide (GSSH) as substrate gave a sigmoidal curve with a Vmax of 122.3 U/mg Universität Bonn, Germany −1 protein, a Kcat of 198 s and a Hill coefficient of 2.3 ± 0.22 suggesting positive Reviewed by: Biswarup Mukhopadhyay, cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly Virginia Tech, United States tetramers. The temperature optimum was 40–45◦C, the melting point 63 ± 1.3◦C in Luyng Xun, thermal unfolding experiments, whereas low activity was measurable up to 95◦C. Site- Washington State University, United States directed mutagenesis showed that residues located in the predicted GSH/GSSH binding *Correspondence: site and in the central hydrogen bond networks including the iron ligands are essential for Arnulf Kletzin activity. Among these, the R139A, D141A, and H171A variants were inactive concomitant [email protected] to a decrease of their melting points by 3–8 K. Other variants were inactivated without Specialty section: significant melting point change. Two out of five cysteines are likewise essential, both This article was submitted to of which lie presumably in close proximity at the surface of the protein (C87 and Microbial Physiology and Metabolism, a section of the journal C224). MalPEG labeling experiments suggests that they form a disulfide bridge. The Frontiers in Microbiology reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide Received: 05 October 2017 and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis Accepted: 27 June 2018 Published: 20 July 2018 and/or protein stabilization. Mass spectrometry revealed modification of C87,C137, Citation: and C224 by 305 mass units equivalent to GSH after incubation with GSSH and with Rühl P, Haas P, Seipel D, Becker J GSH in case of the C87A and C224A variants. The results of this study suggest that and Kletzin A (2018) Persulfide disulfide formation between the two essential surface-exposed cysteines and Cys-S- Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation Residues and Hydrogen Bond and the associated loss of enzyme activity. Networks of the Active Site. Front. Microbiol. 9:1610. Keywords: persulfide dioxygenase, sulfhydryl, sulfur, enzyme kinetics, differential scanning fluorimetry, ETHE1, doi: 10.3389/fmicb.2018.01610 glutathione persulfide, S-glutathionylation Frontiers in Microbiology| www.frontiersin.org 1 July 2018| Volume 9| Article 1610 fmicb-09-01610 July 18, 2018 Time: 16:14 # 2 Rühl et al. Persulfide Dioxygenase From A. caldus INTRODUCTION (4CHL) and the Arabidopsis PDO (2GCU) group in Type I together with many bacterial PDOs including two AcPDOs, the Persulfide dioxygenases (PDOs) catalyze the oxidation of PpPDO and the Myxococcus xanthus enzyme (MxPDO; 4YSB, glutathione persulfide (GSSH) and higher homologs (GSSnH; Figure 1; Pettinati et al., 2015; Sattler et al., 2015). The Type n > 1) with sulfite and reduced glutathione (GSH) as II PDO from Ps. putida is larger than Type I enzymes due products (Tiranti et al., 2009; Kabil and Banerjee, 2012; to additional loops. The Type III enzyme from Staphylococcus Jung et al., 2016). Due to the high reactivity of reduced aureus has a C-terminal rhodanese domain similar to the PpPDO glutathione with elemental sulfur in the enzyme assay (Sluiter, but a different substrate specificity: bacillithiol and coenzyme 1930), PDOs were formerly classified as sulfur dioxygenases A persulfides exceed GSSH and cysteine persulfide 10- to 20- (SDO) because they produce sulfite from GSH-containing fold in terms of their respective specificity constants (Shen sulfur suspensions. SDO activities are known for a long et al., 2015). They all belong to the metallo-b-lactamase (MBL) time from chemolithotrophic sulfur-oxidizing bacteria of the protein superfamily together with glyoxalase II (PFAM database genus (Acidi-) Thiobacillus, however, the protein was neither PF00753). purified successfully nor the corresponding gene identified Several of the amino acid residues, whose mutations cause (Suzuki and Werkman, 1959; Suzuki, 1965a,b; Suzuki and ETHE in humans, are conserved among the PDOs. They are Silver, 1966; Rohwerder and Sand, 2003). More recently, either part of the GSH binding pocket or the extended hydrogen it was found that the human ethylmalonic encephalopathy bond network around the iron site. Some of the latter residues protein 1 (hETHE1) has SDO/PDO activity. hETHE1 plays an coordinate another metal ion in MBL and glyoxalase II, most important role in mitochondrial sulfide detoxification together of which contain dinuclear zinc in the active sites (Cameron with sulfide:quinone oxidoreductase and rhodanese (sulfane et al., 1999; Sattler et al., 2015; for a recent review, see Meini sulfur transferase; Hildebrandt and Grieshaber, 2008; Tiranti et al., 2015). Arg163 is one prominent example among these et al., 2009; Kabil and Banerjee, 2014). Mutations in the ETHE1 residues in hETHE1: mutations here alter protein stability and gene are responsible for the hereditary and fatal autosomal reduction potential of the iron (Figure 1; Tiranti et al., 2006; recessive disorder ethylmalonic encephalopathy (Tiranti et al., Henriques et al., 2014). In spite of the obvious importance, 2004, 2006, 2009; Di Meo et al., 2018), which is characterized no comprehensive mutagenesis study has been published so by high levels of thiosulfate and C3–C5 compounds like far addressing the roles of individual residues in the H-bond ethylmalonic acid in urine and body fluids (Tiranti et al., 2006, network. 2009). Lin et al.(2016) published the first theoretical study recently Triggered by the discovery of ETHE1 in human mitochondria, on the reaction mechanism of the human PDO applying quantum ETHE1-homologous proteins with PDO activity were recently mechanics/molecular mechanics coupled to molecular dynamics identified from Ac. caldus (AcPDO), Ac. ferrooxidans and from calculations. They concluded that “::: the ground state of the several heterotrophic bacteria (Liu et al., 2014; Wang et al., 2014; iron(II)-superoxo reactant is quintet, which can be described as C· · Sattler et al., 2015), sometimes fused with a rhodanese domain GSS -Fe(II)-O2 , and the most feasible reaction channel was (Shen et al., 2015; Motl et al., 2017). The bacterial PDOs seem found to start from the cleavage of dioxygen and a concerted attack to be responsible for the SDO activity observed earlier (Suzuki of distal oxygen on the sulfur atom of the substrate, forming the and Werkman, 1959; Suzuki, 1965a,b; Suzuki and Silver, 1966; metal-bound activated oxygen and a sulfite intermediate.” They Rohwerder and Sand, 2003). also predicted that hETHE1-His81, located in the vicinity of the X-ray structures of the human ETHE1 (PDB identifier 4CHL) iron site and part of a conserved amino acid motif around the and of ETHE1-like PDOs from Arabidopsis thaliana (2CGU) iron ligands (Figure 1; Sattler et al., 2015), plays an important role and several bacteria showed homodimeric or homotetrameric in binding of the persulfide moiety of GSSH. The study did not proteins with molecular masses of 25–30 kDa (or 40–45 kDa include a possible participation of one or more of the cysteines or for the rhodanese fusion proteins; McCoy et al., 2006b; Pettinati the role of the hydrogen bond network around the active site. et al., 2015; Sattler et al., 2015; Motl et al., 2017). The Some of the cysteine residues in PDOs seem to be active sites each contain a mononuclear non-heme iron center important for catalysis and there is evidence that C247 of coordinated by two histidines and one aspartate together with the hETHE1 is essential not only for enzyme activity but three water molecules. The resulting octahedral coordination also for an inferred polysulfidation of other cysteine residues sphere is known as 2-His-1-carboxylate facial triad and is in the protein (Jung et al., 2016). C247 is present in the − typical for over 100 different oxygenases (for a recent review, oxidation state of sulfinic acid (Cys-SO2 ) in the crystal see Kal and Que, 2017). The active site cavity comprises the structures of the hETHE1 and the Arabidopsis enzyme but GSH-binding residues, which position the substrate, so that not in the bacterial proteins (McCoy et al., 2006b; Pettinati the sulfur atom(s) bind to the iron displacing one or more et al., 2015; Sattler et al., 2015; Motl et al., 2017). Inhibition of the water ligands as shown by the 3D structures of the studies of the AcPDO with thiol-binding reagents also indicated PDOs from Pseudomonas putida (4YSL) and Paraburkholderia the importance of the cysteine residues (Wang et al., 2014; phytofirmans (PpPDO; 5VE5; Sattler et al., 2015; Motl et al., Jung et al., 2016). Interestingly, the cysteine residues are 2017). not conserved among the various PDOs and their numbers Persulfide dioxygenases are divided into three subfamilies, vary (Figure 1) so that the function of the cysteine(s) is Type I–III (Liu et al., 2014; Xia et al., 2017).

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