Time Resolved Crystallographic Analysis of Cooperative Ligand Binding and Ligand Migration in a Dimeric Hemoglobin

Abstract

Invertebrate hemoglobins, which range in size from dimers to assemblies of hundreds of subunits, offer excellent models for the investigation of allosteric protein function. A recurring theme among cooperative invertebrate hemoglobins is a subunit pairing involving the heme-embedding E and F helices, despite a lack of sequence conservation in these helices. This is quite different from the assembly of vertebrate hemoglobins and suggests that such a pairing is amenable for regulating oxygen delivery. We have carried out extensive time-resolved crystallographic analysis on the simplest of these hemoglobins, the cooperative homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis. Limited ligand-linked subunit rotation permits the full allosteric transition to be followed in crystals of HbI. These studies have been combined with mutant and functional studies along with conventional crystallographic analysis to provide a comprehensive understanding of the communication between subunits and the movement of ligands through the protein. Investigation of ligand migration through this protein suggests, surprisingly, that ligands primarily escape through a distal histidine gate, despite its involvement in the subunit interface. As a result, ligand escape may require transient subunit movement, which is inhibited in the protein crystal lattice. These findings emphasize the central role of dynamics in protein function. Our studies are also directed towards understanding cooperative protein function in larger invertebrate hemoglobin assemblies. Preliminary time-resolved crystallogaphic analysis on the tetrameric Scapharca HbII suggests similar structural transitions as HbI and offers the possibility of investigating how changes in one subunit directly impact a neighboring subunit in the heterodimeric halves of HbII. In addition, we will discuss new insights that may provide unifying themes for the basis of cooperativity among diverse invertebrate hemoglobins that form EF dimeric pairing.