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

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.


Ratiometric intracellular ATP:ADP measurements.
To measure changes in cellular energy state, we used PercevalHR, a ratiometric intracellular ATP:ADP fluorescent biosensor (3). HepG2 cells were seeded on collagen coated glass bottom dishes (Mattek Corporation) and transfected with a Perceval High Resolution (PercevalHR) reporter plasmid using TransIT®-LT1 reagents (Mirus Bio). GW1-PercevalHR was a gift from Gary Yellen (Addgene plasmid # 49082). A DMI6000 inverted confocal microscope integrated to a Leica TCS-SP5 workstation was used to examine fluorescence signals. The following excitation wavelengths were used to illuminate the fluorophore: 405 nm for and 488 nm. Emissions were recorded with a 20x objective and bandpass filters of 509-578 nm. Fluorescence images were collected every 5 s. The drugs (KCN, 0.1 nM or 10 µM final concentration, iodoacetamide; IAA, 1 mM final concentration) were added to the DPBS solution (Gibco # 14040117). Regions of interest (ROIs) were placed within 1-10 µm along the free edge of the cells. Fluorescence values were calculated after background subtraction (fluorescence intensity of regions without cells). By taking the ratio of the fluorescence at two different excitation wave lengths (Fhigh/Flow), the ratiometric signal reports the occupancy of PercevalHR independent of the amount of sensor protein. Fluorescence intensity ratios were normalized in each experiment to the averaged basal value preceding the treatment period. Individual trajectories and averages were plotted.

Cell proliferation assay.
The cell proliferation ELISA BrdU (colorimetric) assay was performed using Corning Costar TC-Treated flat-bottomed 96-well plates (4). Following pharmacological treatments, cells were incubated with 10 µM BrdU labelling solution for 4 h at 37 o C in a humidified incubator with 5% CO2 and 95% air. The assay is based on the principle that pyridine analogue BrdU incorporates (in place of thymidine) into the newly synthesized DNA strands of proliferating cells. BrdU incorporation was detected by immune-peroxidase staining and a subsequent colorimetric substrate reaction as per the manufacturer's protocol. Plates were read at 450 and 690 nm (reference wavelength) using an Infinite 200 PRO microplate reader. Developed color and absorbance values reflect the amount of DNA synthesis, which correlates with the number of proliferating cells.

Western blotting.
Cells were washed once with ice-cold 1X PBS and harvested in pre-cooled 1X ELISA lysis buffer previously supplemented with protease/phosphatase inhibitor cocktail (1X). Following 2 freeze/thaw cycles, whole-cell lysate was collected, sonicated for 5 min (30 sec ON / 30 sec OFF) in an ultrasonic water-bath, and the total protein was extracted by centrifugation at 16,000 x g at 4 o C for 15 min. The Pierce Coomassie Plus Bradford protein assay was used to quantify the protein concentration of the samples.
For the determination of S-glutathionylation from purified Complex IV or cell homogenates, protein samples were added to Laemmli buffer (in non-reducing conditions) supplemented with 25 mM N-ethyl maleimide (NEM) and incubated for 5 min at 95°C. Proteins were transferred in a PVDF membrane and blocked with milk (5% w/v) supplemented with 25 mM NEM. S-glutathionylation was detected with anti-SSG primary antibody (Virogen; 1:500) then reacted with horseradish-peroxidase-conjugated secondary antibodies (5). Complex IV and cell homogenate signals were normalized with COX-II (Abcam, 1:1000) and actin (Cell Signalling, 1:2000) antibody, respectively.

Cyanide binding to Complex IV (Cytochrome C Oxidase, CCOx).
Complex IV titration, adapted from Jones and colleagues (17) was monitored at room temperature by UV-visible absorption spectroscopy, in an Infinite M200 Pro spectrophotometer (Tecan, Männedof, Switzerland) equipped with magnetic stirrer, using a rubber-cap sealed quartz cuvette. Purified Complex IV from bovine heart (Sigma, 5 µM) in 100 mM potassium phosphate buffer, pH 7.4 supplemented with 0.5 % (w/v) N-dodecyl β maltoside (Buffer A) was reduced with sodium dithionite (15 mM final concentration) and washed through a MiniTrap G-25 column (pre-equilibrated with buffer A). After each KCN addition with gas-tight Hamilton syringes, the solution was stirred for 10 min, and cyanide binding to heme a3 was monitored at 427 nm.

Quantification of free thiols of purified bovine Complex IV.
Free protein thiols were quantitated with the DTNB method (4). Purified Complex IV from bovine heart (Sigma, 1 µM, in Buffer A) was reduced with TCEP (0.5 mM final concentration) and on ice for 1 hour. The excess of TCEP was washed out with a MiniTrap G-25 column, followed by incubation for 1h (room temperature) with 0.1 nM KCN or vehicle. The samples were further washed with a MiniTrap G-25 column and eluted in 500 µl of buffer A. 450 µl of the eluate were transferred in an Eppendorf tube and supplemented with 50 µl of 10 mM DTNB (1 mM final concentration) and incubated for 30 min at RT in the absence of light. The remaining 50 µl was used for the activity assay (see below). Spectra were obtained in an Infinite M200 Pro spectrophotometer (Tecan, Männedof, Switzerland) with a quartz cuvette. 2-nitro-5-thiobenzoic acid anion formation was estimated by analyzing absorbance values at 412 nm (ε412 nm = 14,150 M -1 cm -1 ).

Determination of the specific enzymatic activity of Complex IV.
The activity of purified Complex IV was measured using the colorimetric ELISA Complex IV enzyme activity microplate assay kit (Abcam). Each well of the ELISA plate contained 0.2 µg of protein in 200 µl of assay buffer (provided by the manufacturer) and was incubated with various concentrations of KCN or vehicle at room temperature for 3 hours in the absence of light. Complex IV activity was determined colorimetrically by monitoring the oxidation of reduced cytochrome c by the absorbance change at 550 nm over 2 hours at 30 °C.