Mutation of Residue Phe97 to Leu Disrupts the Central Allosteric Pathway in Scapharca Dimeric Hemoglobin

Animesh Pardanani,Quentin H Gibson,Gianni Colotti,William E Royer

doi:10.1074/jbc.272.20.13171

Abstract

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.

Highlights

The homodimeric hemoglobin (HbI)1 from the blood clam Scapharca inaequivalvis offers a simple model system for studying communication between two chemically identical subunits
The side chain of Phe97 undergoes the largest ligand-linked conformational change in HbI. It is tightly packed in the heme pocket, whereas upon ligand binding it is displaced from the heme pocket into the subunit interface
HbI represents a simple allosteric system that is useful for the investigation of intersubunit communication

Summary

Introduction

The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis offers a simple model system for studying communication between two chemically identical subunits. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins (3). Analysis of structures of this hemoglobin at 1.6 Å for the deoxygenated molecule, 1.4 Å for the CO-liganded form, and 1.7 Å for the oxygenated form has provided a framework for understanding the role of individual side chains and interfacial water in mediating cooperativity (2, 4). Movement of Phe into the subunit interface is coupled with disruption of a well ordered interfacial water cluster and movement of the heme groups deeper within each subunit These effects within a subunit alter the nature of interactions between subunits, which presumably encourages movement of the Phe97Ј side chain (second subunit) into the interface, allowing the second subunit to attain a high oxygen affinity state prior to ligand binding (2). The smaller size of the leucine side chain in these hemoglobins allows it to remain packed in the heme pocket in both liganded and unliganded states

Methods

Results

Conclusion