Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre

Abstract

ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association.

Figure 1. ETHE1-p.Arg163Gln and p.Arg163Trp are folded but have decreased stability.

A. Far-UV CD spectra of purified ETHE1 proteins at 0.1 mg.ml⁻¹ in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Spectra are offset for clarity. B. Molecular model of human ETHE1 highlighting the position of Arg163 in respect to the Fe centre, molecular modeling was made based in the Arabidopsis thaliana ETHE1-like protein structure . C. Thermal denaturation curves of ETHE1 proteins, followed by ellipticity variation at 222 nm from which the fraction of unfolded protein is determined. D. Time course of limited proteolysis experiment at 37°C in 0.1 M Tris-HCl, pH 8.5 monitored by SDS-PAGE. See Materials and Methods for details.

Figure 2. ETHE1-p.Arg163Gln and p.Arg163Trp mutations affect the mononuclear iron site reduction potential.

A. EPR spectra of ETHE1 proteins at 1 mg.ml⁻¹ in 150 mM Tris-HCl pH 7.5 and 300 mM NaCl. B. Redox titrations followed by EPR spectroscopy. Solid and dashed lines are Nernst Curves obtained for the different ETHE1 proteins one-electron reduction processes (n = 1) and the oxidation-reduction potentials are listed in Table 1. EPR spectra were recorded at 9.39 GHz microwave frequency, 2 mW microwave power, 1 mT modulation amplitude, 8 K temperature.