Characterization by NMR of the Heme - Myoglobin Adduct Formed during the Reductive Metabolism of BrCCls COVALENT BONDING OF THE PROXIMAL

’ The reductive debxomination of BrCCls by ferrous deoxymyoglobin Ieads to the covalent bonding of the prosthetic heme to the protein. We have previously shown, by the use of peptide mapping and mass spectrometry, that histidine residue 93 is covalently bound to the heme moiety. In the present study the structure of the heme adduct was more campletely determined by ’H and 13C NMIl techniques. We have found that the ring I vinyl group of the prosthetic heme was altered by the additionof a histidine imidazole nitrogen to the a-carbon and a CCl, moiety to the &carbon. The electronic absorption spectra of the oxidized and reduced states of the altered heme-protein indicated that the heme-iron exists in a bis-histidine-ligated form. Analysis of the crystal sttuckme of native myoglobin suggested that for the altered heme-protein, histidine residues 97 and 64 are ligated to the heme-iron and that residue 97 has replaced the native proximal histidine residue 93. These movements, in effect a “histidine shuffle” at the active site, may be responsible for the enhanced reducing activity of the altered protein.