Identification of the cysteine nitrosylation sites in human endothelial nitric oxide synthase

Abstract

S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that S-nitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975/990, and 1047/1049 as being susceptible to S-nitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

FIG. 1.

The protocol of biotin switch assay that was modified to incorporate peptide mass-mapping experiments for detection of sites of nitrosylation in proteins.

FIG. 2.

(A) The mass spectrum of the peaks eluted during nLC-MS/MS run of a modified eNOS digest eluted at 13.7–14.1 min. Two biotinylated peptides 814.4 Da and +2, and 945.8 and +2 were identified due to a mass shift of 428.3 Da. (B) To confirm the biotinylation, peak 814.4 Da was isolated and MS/MS of the peak was done using data-dependent scan as shown. From the partial sequencing, the peptide was confirmed to be DPRLPPCTLR with a single biotin tag attached to the cysteine residue.

FIG. 3.

MALDI-MS of the tryptic digest of the modified eNOS with HCCA as matrix. Five peaks that have been labeled were identified to be biotinylated with single biotin mass tag.

FIG. 4.

MALDI-MS of the peptides eluted with neutravidin column with α-cyano-4-hydroxycinnamic acid (HCCA) as the matrix. Biotin tags have been cleaved off. Three peaks that have been labeled in bold were identified to be sites of S-nitrosylation in eNOS with GSNO as NO donor.

FIG. 5.

MALDI-MS of the peptides eluted from the neutravidin column with HCCA as matrix. The biotin tags have been cleaved off. The two peaks labeled in bold were identified to be sites of S-nitrosylation in eNOS with SNAP as the NO donor.

FIG. 6.

X-ray structure of the oxygenase domain of human eNOS. The sequence was obtained from the Swissprot protein database. The surface cysteines have been labeled, and the structure is shown as a homodimer.