Hydrogen Bonding and Exchange in Oxymyoglobin

Abstract

Myoglobin (Mb) reversibly binds oxygen in vertebrate muscle. It consists of a polypeptide chain of 153 residues and one heme, and closely resembles one subunit of a hemoglobin (Hb) tetramer. In oxygenated myoglobin (oxyMb), the iron atom is coordinated by four porphyrin nitrogen atoms, N_ϵ of the invariant “proximal” histidine (His 8F), and an oxygen molecule (19). A second histidine (His 7E), the “distal” histidine, lies close to the oxygen, and is found in Mb and Hb of most species. Its function is twofold, to present steric hindrance to linear ligands such as carbon monoxide, while favoring bent ones such as oxygen, and to form a hydrogen bond to bound oxygen, stabilizing the heme-oxygen complex. The latter function was proposed by Pauling (18), but was first observed directly in the work described here. At neutral pH a histidine ring normally has a hydrogen atom bonded to one of the two ring nitrogen atoms. His 7E in Mb has one nitrogen facing the oxygen ligand, and the other facing the surrounding solvent (Figure 1). The position of the hydrogen, and therefore the presence or absence of a hydrogen bond to O₂ can be determined unequivocally only by direct observation using neutron diffraction (20). Neutron protein crystallography on oxyMb also affords a more detailed picture of hydrogen bonding and water structure than that available from x-ray studies, and information on hydrogen exchange.