Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O₂ supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe²⁺) form, indicating a His-Fe²⁺-His binding scheme. O₂ or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe³⁺) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k _on) and a slow dissociation rate (k _off) for both O₂ and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O₂ and CO binding is suggested to be slowin vivo. Because of this competition, the observed O₂ affinity of recombinant human neuroglobin is average (1 torr at 37 °C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 °C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E′_o = −129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSP^WT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, βGlu⁷–βLys¹³², are dependent on which residue of the pair is modified. The βK132A mutants exhibit the spectroscopic signatures of the R → T state transition in both the “hinge” and “switch” regions of the α₁β₂ interface. Both singly and doubly mutated hemoglobin (Hb) βΕ7Α exhibit the switch region signature for the R → T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb βΕ7Α mutants. The observed large decrease in the W3 α14β15 band intensity for doubly mutated Hb βΕ7Α is attributed to an enhanced separation in the A helix-E helix tertiary contact of the β subunits. The results for the Hb A βGlu⁷–βLys¹³² salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the α₁β₂ dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.