A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation

Abstract

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H₂S to glutathione persulfide with concomitant reduction of CoQ₁₀. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ₁-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates ²⁰¹Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and ³⁷⁹Cys-SSH, which transfers sulfur to an external acceptor.

Keywords: butyrate; coenzyme Q; crystal structure; enzyme kinetics; flavin; hydrogen sulfide.

Figure 1.. Intersection between sulfide and butyrate oxidation pathways and the proposed mechanisms for the SQR reaction.

(A) The canonical SQR reaction utilizes GSH as an acceptor to generate GSSH, with electrons driven into the CoQ₁₀ (Q) pool. ACADS via the electron transferring flavoprotein (ETF), also drives electrons into the Q pool via oxidation of butyrate. We hypothesized that SQR utilizes CoA as an alternate acceptor generating CoA-SSH, which inhibits ACADS and blocks electron flow from butyrate oxidation. PDO (or ETHE1), TST and SO represent persulfide dioxygenase, rhodanese and sulfite oxidase, respectively in the mitochondrial sulfide oxidation pathway. (B) The previously proposed SQR reaction mechanism in which the active site cysteines in the resting enzyme form a disulfide. Cys201 participates in a CT complex with FAD while Cys379 is the sulfane sulfur carrier. (C) A proposal for the SQR-catalyzed oxidative half reaction, informed by our crystal structures, invokes a trisulfide configuration of the active site cysteines. Sulfide addition leads to persulfides on both cysteines. ²⁰¹Cys-SS⁻ forms a CT complex with FAD while ³⁷⁹Cys-SSH serves as the sulfur donor.

Figure 2.. Pre-steady state kinetic analysis of ndSQR-mediated sulfur transfer to CoA.

(A) Dependence of ndSQR activity on CoA concentration assessed in the coupled assay described under Star Methods. The data represent the mean ± S.D. of three independent experiments, each performed in duplicate. (B) ndSQR (40 μM) in 100 mM potassium phosphate buffer, pH 7.4, was rapidly mixed 1:1 (v/v) with Na₂S (80 M) for ~35 msec to form the sulfide-induced CT complex at 675 nm (dashed black line), followed by a second rapid 1:1 (v/v) mixing with CoA (3 mM). Spectral changes associated with CT complex decay in ndSQR and concomitant FAD reduction (red line) were monitored over a period of 3 sec. (C) Comparison of the decay kinetics of the sulfide-induced CT complex in ndSQR with or without mixing with CoA (3 mM) monitored at 675 nm over 3 sec. (D) Dependence of the k_obs for sulfide-induced CT complex decay on CoA concentration. The data represent the mean ± S.D. of two independent experiments.

Figure 3.. CT complex formation in ACADS by ndSQR-derived CoA-SSH.

(A) ACADS (10 μM) in 100 mM potassium phosphate buffer, pH 7.4 at 25 °C (black line) was mixed with CoA-SSH (20 μM) and incubated for 1 min. Formation of the CoA-SSH-induced CT complex in ACADS was indicated by the 710 nm absorbance band (red line). (B) ACADS (10 μM) in the same buffer as in (A), was mixed with Na₂S (150 μM), CoA (3 mM), and CoQ₁ (60 μM) and incubated for 1 min (black line), followed by addition of ndSQR (100 nM) and incubation for 1 min. Formation of the CoA-SSH-induced CT complex in ACADS (red line) was observed. The data are representative of three independent experiments.

Figure 4.. Overall structure and active site architecture of SQR.

(A) Overall structure of SQR-CoQ₁ in which the monomers in the crystallographic dimer are shown in pale green and pink, respectively. The C terminal membrane anchoring helices are highlighted in cyan and salmon pink. FAD and CoQ₁ are shown in stick display. The electron density maps (2F_o-F_c) contoured at 1.0 σ of Cys379, Cys201, FAD and CoQ₁ are shown as mesh in SQR-CoQ₁ (B), SQR-CoQ₁ + sulfite (C) and SQR-CoQ₁ + sulfide (D). Note that the cysteine backbone carbons are shown in green in B-D and that CoQ₁ has moved away from FAD in the sulfide-soaked crystal structure in (D). (E-G) Close up of the active site redox centers in the indicated structures in which the distances between sulfur atoms and between sulfur atoms and the flavin C4a are labeled. The diffraction data precision indicator values are 0.37 Å (E), 0.16 Å (F), and 0.28 Å (G), respectively.

Figure 5.. The sulfur substrate entry site is on the matrix side.

(A) Electrostatic potential energy surface map showing the sulfur substrate entry/exit site in SQR-CoQ (dashed box and arrow, left panel). In the right panel, the structure is shown in the same orientation as in the left panel, with the FAD and CoQ₁ displayed as yellow and dark gray spheres, respectively. (B) Docking model of SQR-CoQ₁ with CoA (left) and close-up view of the active site (right). (C) Docking model of SQR-CoQ₁ with GSH (left) and close-up view on the active site (right).

Figure 6.. CoQ entry from the mitochondrial membrane side.

(A) Electrostatic potential energy surface map showing the CoQ entry/exit site denoted by the dashed line in the SQR-CoQ₁ structure (left panel). In the right panel, the structure is shown in the same orientation as the left panel, with FAD and CoQ₁ displayed as yellow and grey spheres, respectively. (B) A close-up showing the interactions between CoQ₁ and active site residues. (C) Location of CoQ₁ near FAD at the end of the hydrophobic tunnel (in mesh representation) as seen in the SQR-CoQ₁ structure (green) versus at the entrance to the tunnel (cyan) in the SQR-CoQ₁ + sulfide structure.