Abstract
Hydrogen sulfide (H2S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron-containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH. PDO mutations result in the autosomal-recessive disorder ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH), in which the cysteinyl moiety in GSH is substituted with homocysteine, as a mechanism-based PDO inhibitor. Human PDO used GHcySH as an alternative substrate and converted it to GHcy-SO2H, mimicking GS-SO2H, the putative oxygenated intermediate formed with the natural substrate. Because GHcy-SO2H contains a C-S bond rather than an S-S bond in GS-SO2H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y, and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-11-fold) and lower thermal stability (1.2-1.7-fold) than WT PDO. They also exhibited varying degrees of catalytic impairment; the kcat/Km values for R163W, L55P, and C161Y PDOs were 18-, 42-, and 65-fold lower, respectively, and the T136A variant was most affected, with a 200-fold lower kcat/Km Like WT enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.
Highlights
Hydrogen sulfide (H2S) is a signaling molecule with many beneficial effects
Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron– containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH
We show that GHcySH undergoes dioxygenation, forming GHcy-SO2H, which remains bound and inhibits PDO
Summary
The mutant proteins were purified using a one-step protocol and were Ͼ95% pure, which is comparable with the purity obtained for WT PDO (not shown). The mutants were less stable and exhibited Tm values of 47 Ϯ 6 °C (L55P), 44 Ϯ 2 °C (T136A), 38 Ϯ 3 °C (C161Y), and 53 Ϯ 2 °C (R163W) (Table 1). The mutants exhibited between 9 and 71% of the ferrous iron content of the WT enzyme (Table 1). The rate of oxygen consumption during enzyme-catalyzed oxygenation of GSSH (Reaction 1) was monitored as a measure of PDO activity. The mutants displayed varying degrees of catalytic impairment with specific activities ranging from 1.5 to 11% of WT PDO (Table 1), which was independent of their metal ion content. The mutants exhibited modest increases (ϳ2–3-fold) in the Km value for GSSH (Fig. 3), yielding kcat/Km values that were 18 –200-fold lower than WT PDO (Table 1). The kinetic parameters were determined by monitoring O2 consumption in the presence of GSSH at 22 °C in 100 mM sodium phosphate, pH 7.4, with 0.5–170 g of enzyme as described under “Experimental procedures.”
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