Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals

Area of Science:

• Biochemistry
• Structural Biology
• Oxygen Transport

Background:

• Hemoglobin (Hb) exhibits cooperativity in oxygen binding, a phenomenon typically explained by conformational changes between T (tense) and R (relaxed) states.
• Investigating T-state Hb's intrinsic oxygen binding properties is crucial for understanding allosteric regulation.
• Metal hybrid hemoglobins offer a method to separately assess the oxygen affinities of alpha and beta subunits.

Purpose of the Study:

• To determine the three-dimensional (3D) structure and oxygen binding characteristics of alpha(Fe2+)2beta(Ni2+)2 crystals.
• To investigate the oxygen affinity of alpha and beta subunits independently in T-state hemoglobin.
• To confirm the noncooperative nature of oxygen binding in the T-state.

Main Methods:

• Crystallization of alpha(Fe2+)2beta(Ni2+)2 hemoglobin from polyethylene glycol solutions.
• Single crystal microspectrophotometry using polarized light to measure absorption spectra at varying oxygen pressures.
• Analysis of binding curves and Hill coefficients to determine oxygen affinity and cooperativity.

Main Results:

• Oxygen binding to alpha(Fe2+)2beta(Ni2+)2 crystals showed Hill coefficients close to unity (0.96 and 0.90), indicating noncooperativity.
• Analysis revealed inequivalent oxygen affinities between alpha1 and alpha2 subunits (1.3-fold difference) and between alpha and beta subunits (1.2-fold difference).
• The observed heterogeneity in subunit affinities, in the absence of cooperativity, accurately predicted the overall noncooperative binding curve of Hb A.

Conclusions:

• Oxygen binding by T-state hemoglobin is intrinsically noncooperative, consistent with the Monod, Wyman, and Changeux model.
• Subunit inequivalence in oxygen affinity exists within the T-state crystal lattice due to packing interactions.
• Cooperativity in hemoglobin function arises from mechanisms beyond the intrinsic binding properties of the T-state.