Ligand Diffusion Pathways and Mechanisms for Regulating Oxygen Affinity in Two Model Invertebrate Globins: The E7 Gate and Apolar Tunnel

The major pathway for O 2 binding to mammalian myoglobins (Mbs) and hemoglobins (Hbs) involves transient outward movements of the distal histidine (HisE7), which allows ligand migration into the distal portion of the heme pocket. This E7 gate pathway appears dominant in vertebrate Hbs and Mbs. However, a number of invertebrate globins, including the dimeric hemoglobin from the blood clam Scapharca inaequivalvis (ScHbI), have an inverted quaternary structure in which an EF:FE dimer interface appears to block the HisE7 gate. Another set of globins, including the mini-hemoglobin from the Nemertean sea worm Cerebratulus lacteus (CerHb), are missing the N-terminal A-helix, which results in an internal tunnel between the E- and H-helices. This apolar channel has been suggested to represent an alternative to the HisE7 gate pathway. To determine the roles of the E7 gate and alternative pathways, we have systematically examined the effects of mutations at the E7 position in ScHbI and CerHb and at 21 other positions along the polar channel in CerHb. As was observed for SwMb and HbA, there is a progressive decrease in the bimolecular rate constants for O 2 binding to ScHbI as the size of the amino acid at position E7 is increased from Ala to Trp. This pattern is unaffected when ScHbI is completely converted to the R- or high affinity quaternary state by the F97Y mutation or when the dimer interface is completely disrupted by the K30D mutation. In contrast, E7 mutations have little affect on the rates of ligand entry and escape in CerHb. Instead, ligands diffuse through the apolar channel between the E- and H-helices as judged by decreases in both overall association and dissociation rate constants and increases in the extent of geminate recombination when the channel is blocked by small to large amino acid mutations. In SwMb, these trends are only observed when the small to large mutations are constructed at or near the E7 gate or directly in the distal pocket where ligands are captured. Thus, it is clear that globins have evolved more than one pathway for rapid O 2 uptake and release.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Natural Sciences

Grantor

Rice University

Year dc:date.issued

2011

Author dc:creator

• Salter, Mallory D.

Advisor dc:contributor.advisor

• Olson, John S.

• Pure sciences
• Hemoglobin
• Ligand diffusion
• Oxygen binding
• Biochemistry

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/70423

OAI identifier oai:identifier

oai:repository.rice.edu:1911/70423