Gayathri Vasudevan

Ordered heme binding ensures the assembly of fully functional hemoglobin: a hypothesis.

The exact mechanism by which four Fe-Protoporphyrin-IX (heme) moieties and four nascent globin chains combine to form human hemoglobin (alpha(2)beta(2)) remains a mystery. Recent Soret spectral static and kinetic studies of the incorporation of CN-Hemin derivatives into an array of human globin species have provided in vitro evidence of an ordered assembly pathway, through an alphaheme-betaglobin intermediate, that ensures correct formation of active hemoglobin tetramers.

Wavelength-Dependent Spectral Changes Accompany CN-Hemin Binding to Human Apohemoglobin

The interaction of apohemoglobin with two heme derivatives, CN-protohemin and CN-deuterohemin, was monitored at multiple Soret wavelengths (417–423 and 406–412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10°C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both CN-protohemin and CN-deuterohemin derivatives with the a chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of α- and β-chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral a propionyl 45(CE3); 58(E7); 61(E10) as well as the b vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the αβ dimer interface.

Analysis of the Global Architecture of Hemoglobin A2 by Heme Binding Studies and Molecular Modeling

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A2 was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5−13 × 107 M−1 s−1), local structural rearrangement (0.21−0.23 s−1), global αδ structural event (0.071−0.098 s−1), and formation of the Fe–His bond (0.009−0.012 s−1), respectively. Kinetic differences observed between apohemoglobin A2 and apohemoglobin A (previously studied) prompted an analysis of the structures of β and δ chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.

Soret Spectroscopic and Molecular Graphic Analysis of Human Semi-β-Hemoglobin Formation

The interaction of heme-free α (αo) and heme-containing β (βh) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 107 M−1 s−1. This was 100-fold more rapid than the reported rate of combination of αh and βh proteins. This encounter-like rate of semi-β-hemoglobin (αoβh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled αo protein superimposed over native αh protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the α1β1 interface, was apparently responsible for the enhanced association properties of the αo protein.