Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics

Abstract

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H₂S and may be considered the fourth mammalian gasotransmitter.

Keywords: bioenergetics; gasotransmitters; mitochondria.

Fig. 1.

(A–C) Low cyanide concentrations stimulate, while high cyanide concentrations suppress mitochondrial electron transport, CCOx activity, and ATP biosynthesis in HepG2 cells, as measured by extracellular flux analysis (A and B) or a fluorescent ATP:ADP biosensor (C). KCN in A and C was applied at 0.1 nM (low concentration) or 10 µM (high concentration). In C, after KCN (0.1 nM or 10 µM) the GAPDH inhibitor iodoacetamide (IAA; 1 mM) was applied, which produced a rapid decrease in ATP levels. In B, a broad range of cyanide concentrations was tested (0.1 nM to 10 mM). (D) Cyanide (0.1 nM, 6 h) increases proliferation in various human cell lines. (E) High cyanide concentrations (10 µM, 6 h), especially after pretreatment of the cells with the glycolysis inhibitor 2-deoxyglucose (2DG; 5 mM, 2h), suppress HepG2 cell proliferation. (F) Cyanide exposure (0.1 nM or 10 µM, 6 h) does not affect mitochondrial complex expression levels in HepG2 cells. (G) Cyanide (0.1 nM) catalyzes constitutive S-glutathionylation removal from purified CCOx, evidenced by western blotting (30- and 57-kDa subunits; G) and the DTNB assay (H); these effects are associated with an increase in CCOx-specific activity (I). (J) Cyanide (0.1 nM to 100 µM) induces spectral changes in CCOx. (K and L) Pharmacological suppression of cellular glutathione levels with buthionine sulfoximine (BSO; 0.5 mM, 24 h) increases CCOx activity in HepG2 cells and reduces protein glutathionylation in whole-cell lysates. (M) HepG2 cells exposed to diluted supernatants (DSNs) of P. aeruginosa (PAO1; cyanide: ∼20 nM) stimulate CCOx activity, while concentrated supernatants (CSNs; cyanide: ∼2 µM) inhibit CCOx; these effects were absent when supernatants of an HcnC-KO Pseudomonas (a mutant strain, deficient in cyanide production) were used (HcnC KO). Control: CTR; oxygen consumption rate: OCR; phosphate-buffered saline: PBS; glyceraldehyde 3-phosphate dehydrogenase: GAPDH; relative fluorescence unit: RFU; UWQCRC2: cytochrome B-C1 complex subunit 2, (mitochondrial); SDHB: succinate dehydrogenase complex iron sulfur subunit B; COX II: cytochrome C oxidase subunit II; NDUFB8: NADH:Ubiquinone oxidoreductase subunit B8; DTNB: 5,5'-dithiobis-(2-nitrobenzoic acid). *P < 0.05; **P < 0.01.

Fig. 2.

Expression of epoxide hydrolase (EPHX), β-glucosidase (GBA), myeloperoxidase (MPO), methylmalonic aciduria and homocystinuria type C protein (MMACHC), 3-mercaptopyruvate sulfurtransferase (3-MST), and thiosulfate sulfurtransferase (TST) in HepG2 cells under baseline conditions and after exposure to various concentrations of cyanide for 6 h. β-actin was used as loading control. (A) Representative western blots; (B) densitometry analysis. Note the biphasic changes in MPO expression in response to cyanide. CTR: control conditions, in the absence of added cyanide. *P < 0.05; **P < 0.01.