Direct evidence for S-nitrosation of mitochondrial complex I

Abstract

NO* (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO* to the Cu(B)/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 microM-1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.

Figure 1. Chemiluminescent analysis of SNO

Upper panel shows a schematic of the derivatization chemistry. Each sample was divided into three aliquots. Sulphanilamide reacts with NO₂⁻, HgCl₂ reacts with SNO. The lower panel shows a typical chemiluminescent NO analyser (NOA) trace, for injection of samples derived from GSNO-treated isolated rat heart mitochndria.

Figure 2. Characterization of mitochondrial S-nitrosation

(A) S-nitrosation pattern of rat heart mitochondria, visualized by biotin-switch assay, following treatment with various NO^• donors as described in the Materials and methods section. Molecular mass markers (kDa) are shown to the right of the blot. (B) Dose response to GSNO. Mitochondria were treated with various doses of GSNO and analysed as described for (A). (C) SNO content analysed by chemiluminescence. Mitochondria were treated with various doses of GSNO then analysed by chemiluminescence as detailed in the Materials and methods section. (D) NO₂⁻ content analysed by chemiluminescence, as described for SNO in (C). In (C) and (D), axes are broken between 500 μM and 1 mM GSNO to enhance detail at lower concentrations. Results are expressed as the means±S.E.M. of at least 3 independent experiments. Ctrl, control; mito′, mitochondrial.

Figure 3. Blue-native gel electrophoresis and chemiluminescent SNO analysis

(A) Separation of the mitochondrial respiratory complexes using blue-native gel electrophoresis. The position of the complexes is shown by Roman numerals to the right, and the percentage of total distance down the gel (relative to the dye front) is shown to the left. (B) Chemiluminescent SNO scan of proteins extracted from 2 mm segments of the blue native gel. Each point represents the amount of SNO detected in a given 2 mm gel slice. SNO content data are means of 2 determinations, and overall the data are representative of at least 3 independent experiments.

Figure 4. Inhibition of complex I following GSNO treatment

(A) Complex I activity was monitored in mitochondria treated with 1 mM GSNO. For ‘dark’ samples the entire experiment including the assay was performed in the dark. For ‘light’ samples the GSNO treatment was in the dark, but the mitochondrial pellet was exposed to an intense white light source prior to the complex I assay. (B) Complex I activity was monitored using mitochondria treated with 10–500 μM GSNO. Mitochondria from the same suspension were carried over to blue-native gel electrophoresis followed by chemiluminescent SNO detection. Stoichiometric quantification of S-nitrosation was calculated as detailed in the text. (C) Percentage complex I inhibition was plotted against complex I SNO. The line fit and r² value were obtained by regression analysis.

Figure 5. Superose 6 column chromatography and chemiluminescent SNO analysis

(A) Profile of the protein content in each fraction collected from the column. The relative position of each fraction in the collection profile is expressed as a percentage of the total column bed volume. (B) Chemiluminescent SNO scan of the column fractions. (C) Plot of protein content against SNO content of column fractions. The fit line and r² value are for a linear regression analysis. (D) Complex I activity of the column fractions. All results are representative of at least three independent experiments. Abbreviation: NOA, NO analyser.

Figure 6. Biotin-switch analysis of SNO-enriched peak 1

(A) Coomassie Blue stained gel, comparing the proteins found in a total mitochondrial protein extract with those found in SNO peak 1 from Superose 6 column chromatography (Figure 5B). (B) Streptavidin–horseradish peroxidase Western blot of SNO peak 1 from Superose 6 column chromatography (Figure 5B) subjected to biotin-switch derivatization. Results are shown for control and GSNO-treated mitochondria, and are representative of at least three independent experiments. Mito′, mitochondrial; Ctrl, control.

Figure 7. SNO detection in IPC

Rat hearts were subjected to IPC, mitochondria isolated, and the amount of mitochondrial SNO measured using chemiluminescent analysis, all in the dark, as detailed in the Materials and methods section. N.D., not detectable (limit of detection 0.5–1 pmol of SNO).