Horse heart mini- and full length-myoglobin: pH effects on CO binding.

Horse heart mini-myoglobin, a fragment of full-length myoglobin, binds carbon monoxide (CO) differently depending on pH, revealing distinct conformations that affect its ligand binding and release. This suggests key roles for protein regions outside the fragment in oxygen delivery to tissues like the retina.

Introduction and Background

• Myoglobin (Mb): A protein in muscle tissues that binds oxygen and facilitates its delivery.
• Horse heart myoglobin (HH-Mb): The full-length muscle protein used as a model for studying ligand binding kinetics.
• Mini-myoglobin (mini-HH-Mb): A proteolytic fragment comprising residues 32-139, corresponding roughly to the central exon of the gene.
• Context: Investigates how the truncated mini-myoglobin binds CO compared to the full-length HH-Mb, particularly how pH affects this binding.

Key Experimental Observations

• Kinetics of CO Association:
  • HH-Mb shows a single exponential kinetic pattern for CO binding and release, indicating a single dominant conformation.
  • Mini-HH-Mb shows biphasic kinetics for both CO association and dissociation, implying existence of at least two distinct conformations in slow or no equilibrium.
• pH Dependence on CO Binding:
  • For mini-HH-Mb, CO association reveals two proton-linked transitions:
    • One transition in the neutral-alkaline range, unique to mini-HH-Mb.
    • One transition in the acidic range, with a pK ~ 2.9, similar to HH-Mb (pK ~ 2.7).
  • CO dissociation from mini-HH-Mb was studied between pH 5.5 and 10.5 because outside this range protein denaturation altered kinetics.
  • CO dissociation rates for mini-HH-Mb have a bell-shaped pH dependence, meaning rates vary with pH and peak at some optimal point.
  • In contrast, HH-Mb CO dissociation rate is pH-independent.

Structural and Functional Interpretations

• Molecular Modeling Results:
  • Mini-HH-Mb shows increased flexibility around the heme pocket, especially on the proximal and distal sides that bind the heme iron.
  • This flexibility can explain the multiple conformations and kinetic behaviors observed experimentally.
• Role of N- and C-Terminal Regions:
  • The missing terminal regions in mini-HH-Mb appear to stabilize conformations and affect ligand binding/release properties.
  • This suggests these terminal parts play a significant role in oxygen binding affinity and kinetics in full-length protein.

Physiological Implications

• Oxygen Supply to Tissues:
  • HH-Mb’s precise oxygen binding and release kinetics are crucial for tissues with high oxygen demand, such as the retina.
  • The differences in CO binding kinetics in mini-HH-Mb imply the full-length protein’s structure fine-tunes oxygen delivery efficiency.
• Potential Therapeutic Applications:
  • Understanding these differences could help develop strategies to improve retinal blood flow in eye diseases where oxygen delivery is impaired.

Summary

• The study compares full-length horse heart myoglobin and its proteolytic mini-fragment in terms of carbon monoxide binding under varying pH conditions.
• Mini-HH-Mb exhibits two slow-equilibrating conformations, leading to biphasic ligand kinetics, not seen in the full-length protein.
• pH affects the CO binding differently in mini-HH-Mb, revealing proton-linked transitions absent in full-length HH-Mb.
• Structural modeling suggests increased flexibility in mini-HH-Mb’s heme pocket underlies these kinetic differences.
• The N- and C-terminal regions absent in mini-HH-Mb play a critical role in maintaining the full myoglobin’s function, relevant for oxygen transport in demanding tissues such as the retina.