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

Abstract

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 (V_max) at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. K_M for GSH and K_cat were 0.5 mM and 181 s^-1, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a V_max of 122.3 U/mg protein, a K_cat of 198 s^-1 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 R₁₃₉A, D₁₄₁A, and H₁₇₁A 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 (C₈₇ and C₂₂₄). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C₈₇, C₁₃₇, and C₂₂₄ by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the C₈₇A and C₂₂₄A 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.

Keywords: ETHE1; S-glutathionylation; differential scanning fluorimetry; enzyme kinetics; glutathione persulfide; persulfide dioxygenase; sulfhydryl; sulfur.

FIGURE 1

Multiple alignment of iron-containing persulfide dioxygenase (PDO) sequences. Sequences were downloaded from GenBank, aligned using the MAFFT server and manually corrected with respect to the 3D structures (GenBank/PDB accession in species names). Type I, Type II, and Type III enzymes were grouped according to Xia et al. (2017). Bars, alpha helices; arrows: beta sheets; |, residues correlated with ETHE according to Refs. (Tiranti et al., 2004, 2006; Jung et al., 2016); +, iron ligands; #, cysteine residues at the surface of the MxPDO (Sattler et al., 2015); , other cysteine residues of the AcPDO; • secondary coordination sphere and hydrogen bonding network residues; black ↑, GSH-binding residues in all PDOs; blue ↑, GSH-binding residues in all PDOs; gray ↑, GSH-binding residues in Type II PDOs.

FIGURE 2

Temperature (A) and pH profiles (B) of the AcPDO with 0.2 mM GSH and 2% sulfur and various amounts of wild type AcPDO as appropriate (1–100 μg/ml). Error bars represent the standard deviation from triplicate measurements.

FIGURE 3

Enzyme kinetics of the AcPDO. (A) activity vs. substrate concentration plot for GSH plus sulfur and GSSH; Error bars represent the standard deviations from triplicate measurements. (B) Lineweaver–Burk plot for GSH plus sulfur; (C) Hill plot for GSSH.

FIGURE 4

Native AcPDO and effects of denaturants. (A) Non-denaturing 4–16% polyacrylamide gel with different amounts of AcPDO; M, Native marker liquid mix for BN/CN (Serva, Heidelberg, Germany); (B) Gel permeation chromatography of freshly prepared AcPDO (0.5 mg protein) with marker proteins; (C) Gel permeation chromatography of previously frozen AcPDO (0.1 mg protein) in the presence of different concentrations of guanidinium hydrochloride.

FIGURE 5

Molecular representations of the AcPDO 3D model with glutathione from the PpPDO structure (5VE5). (A) Theoretical model of GSH in the AcPDO active site structure and predicted hydrogen bonds; boldface amino acid residues were mutagenized in this study. (B) C₈₆ and C₂₂₃ in the MxPDO 3D structure (gray) and in the AcPDO model (green). (C) Comparison of the secondary coordination sphere around D_129/130 between the MxPDO (gray) and the AcPDO (green) originating from the S₁₁₆ residue in the MxPDO.

FIGURE 6

Effects of site-directed mutagenesis in the AcPDO gene on the enzyme activity and iron content of the corresponding protein variants. (A) Variants of the iron ligands. (B) Reconstitution of the H₅₇G variant with Fe and imidazole in the enzyme assay buffer. (C) Reconstitution of the H₁₁₃G variant with Fe and increasing concentrations of imidazole. (D) Mutagenesis of substrate-binding site and hydrogen bond network. (E) Cysteine variants. Error bars represent the standard deviation from triplicate measurements.

FIGURE 7

Analysis of AcPDO using a MalPEG gel shift assay. (A) Coomassie-stained 10% Tris-tricine gel of the AcPDO wild type (10 μg/lane). (B) Western analysis using StrepMAP-Classic HRP-conjugated antibody. M, Marker in kiloDalton, NEM, sample derivatized with N-ethylmaleimide, DTT, sample reduced with dithiothreitol.

FIGURE 8

ESI mass spectra of the AcPDO wild type, the C₈₇A and the C₂₂₄A variants of the as-isolated proteins and after incubation with GSH and GSSH, respectively.

FIGURE 9

Effect of Inhibitors on the AcPDO. (A) Residual PDO activity with 1 mM GSH, sulfur and N-Ethylmaleimide (NEM) or iodoacetamide (IAA) after adding the substance directly to the enzyme assay mixture. (B) Same as in A only that the GSH concentration was varied. (C) Residual PDO activity after pre-incubation of the AcPDO with NEM, IAA, or Tris(2-carboxyethyl)phosphine (TCEP). Error bars represent the standard deviation from triplicate measurements.

FIGURE 10

First derivative plots of differential scanning fluorimetry (tryptophane/tyrosine fluorescence) of AcPDO variants (1 mg/ml) measured in 100 mM Tris buffer, pH 8.0, at a heating rate of 1 K/min and comparison with wild type.