Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins.

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO-liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 A resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas complete absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF-dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand-induced structural changes of Oligobrachia Hb are quite different from those of the well-studied mollusk Hb having the same EF-dimer structure. The structural rearrangements make the dodecameric subassembly form a tighter conformation than those of fully oxygenated or CO-liganded dodecamer structure.