SOLUTION OF H-1-NMR STRUCTURE OF THE HEME CAVITY IN THE OXYGEN-AVID MYOGLOBIN FROM THE TREMATODE PARAMPHISTOMUM-EPICLITUM

A two-dimensional H-1 NMR study has been carried out on the heme cavit y of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr , and 2 upfield ring current aliphatic side chains. Based solely on th e conserved myoglobin folding topology that places the E helix-heme cr ossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix al ignment were unambiguously established that place Tyr(66) at position E7. Moreover, all eight aromatic and the two aliphatic side chains wer e shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr(32)(B10) and Tyr(66)(E7) rings indicate t hat both residues are oriented into the heme cavity, which is unpreced ented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordina ted oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autox idation resistance of this myoglobin.