SPECIFIC AZIDOPHENYLDIAZENE HEMOPROTEIN ACTIVE-SITE PROBES - CROSS-LINKING OF THE HEME TO HIS-64 IN MYOGLOBIN

The reaction of myoglobin (Mb) with (meta- and (para-azidophenyl)diaze ne yields the corresponding sigma-bonded meta- or para-phenyl-iron com plex. Aerobic denaturation of these complexes in the dark yields N-(az idophenyl)protoporphyrin IX adducts. Photolysis of either the meta- or para-azidophenyl-iron complex of Mb prior to denaturation results in cross-linking of the protein to the prosthetic group via a linker form ally composed of a phenyl group and a nitrogen atom. Tryptic digestion of the modified protein and mass spectrometric analysis of the peptid es identifies His-64 as the residue to which the heme is attached by b oth probes. Photolysis of the azidophenyl-iron complexes is postulated to produce an iron-bound arylnitrene that binds directly, or after re arrangement to a seven-membered cyclic ketenimine, to the protein. Sub sequent shift of the aryl group from the iron to a porphyrin nitrogen generates the heme-protein cross-link. This approach unambiguously ide ntifies hemoprotein active site residues and defines their location wi th respect to the heme iron atom. This approach should prove useful in characterizing the active sites of structurally undefined hemoprotein s because aryl-iron complex formation is a general hemoprotein reactio n.