Abstract
The photoactivatable trifluoromethyldiazirinylphenyldiazene probes 1a and 2a have been synthesized, and their utility in the mapping of hemoprotein active sites has been validated with myoglobin (Mb). Reaction of the probes with Mb yields Fe-aryl adducts. Photolysis of these adducts unmasks a carbene that cross-links to active-site protein residues. Migration of the aryl group from the iron to a porphyrin nitrogen then attaches the porphyrin chromophore to the labeled amino acid residue. Tryptic digestion of the labeled proteins followed by mass spectrometric analysis of the peptides has identified Leu-29, His-64, Ile-107, and Val-68 as active-site residues. Previous studies with an arylnitrene probe, which appears to only react with nucleophilic groups, identified His-64 as an active-site residue [Tschirret-Guth, R. A.; Medzihradszky, K. F.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1998, 120, 7404−7410]. These studies have identified all but one of the active-site amino acids in contact with the probe. The present strategy not only labels active-site amino acids but also, because the probe is rigidly held in the active site, provides approximate locations for the labeled residues with respect to the heme iron atom. Validation of the strategy with myoglobin opens the way to use of the approach with hemoproteins of unknown active-site structure.
Published Version
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