Direct Observation of Nitrosylated Heme in Myoglobin and Hemoglobin by Electrospray Ionization Mass Spectrometry

Using electrospray ionization mass spectrometry (ESI-MS), we demonstrate the direct observation of NO attached to the heme moiety in horse heart myoglobin (Mb) and in the Rand â-chains of human hemoglobin (Hb). It was found that a narrow range of ESI-MS conditions conspire to make observation of Fe-NO interactions challenging, and this is presumably the reason why earlier attempts by other research groups to detect intact Fe-NO products by mass spectrometry were unsuccessful. For Mb and Hb, mass shifts are observed that are consistent with NO modification of the hemoproteins. ESI mass spectra of the apoprotein portions of Mb and Hb in the presence of NO demonstrated the absence of NO modification of the polypeptide backbones. UV/vis spectra of both Mb/NO and Hb/NO solutions, recorded at the time of ESI-MS analysis, demonstrated hemoproteinII-NO formation. To test the hypothesis that intact nitrosylated heme groups are observable by ESI-MS, a nitrosylated model metalloporphyrin was studied. The ESI mass spectrum of nitrosyl-R,â,γ,δ-tetraphenylporphinatoiron(II), [Fe(TPP)NO], showed peaks that were ascribed to [Fe(TPP)]+ and [Fe(TPP)NO]+. To test further our hypothesis that the hemoprotein-NO peaks are due to heme nitrosylation and contain no significant contributions from NO modification of the polypeptide backbone, we determined the ESI-MS conditions necessary for observing S-nitrosation of Cys residues in Hb. Human Hb contains one Cys residue in HbR (Cys 104) and two Cys residues in Hbâ, but only Hbâ Cys 93 is surface accessible. When metHb was incubated with S-nitroso-N-acetyl-DL-penicillamine (SNAP), the ESI mass spectrum revealed a single SNAP modification in both Hbâ and apoHbâ. The ESI-MS conditions used for analyzing the Hb/SNAP solution were too harsh for observing intact heme nitrosylation, and thus, we ascribe the SNAP-modified Hbâ and apoHbâ peaks to S-nitrosation of Cys 93 in Hbâ. Under appropriate denaturing sample conditions, it proved possible to S-nitrosate all three Cys residues in human apoHb. Our findings demonstrate that (once correct conditions are established) ESI-MS is a powerful tool for the detection of intact Fe-NO interactions in proteins and porphyrins.