Oxygenation Markedly Alters the Quaternary Structure of Hb

Crystals of deoxyhemoglobin shatter when exposed to O2. Further, X-ray crys-tallographic analysis reveals that oxy- and deoxyhemoglobin differ markedly in quaternary structure. In particular, specific a/3-subunit interactions change. The a/ contacts are of two kinds. The a1/1 and a2/2 contacts involve helices B, G, and H and the GH corner. These contacts are extensive and important to subunit packing; they remain unchanged when hemoglobin goes from its deoxy to its oxy form. The a1/2 and a2/1 contacts are called sliding contacts. They principally involve helices C and G and the FG corner (Figure 15.30). When hemoglobin undergoes a conformational change as a result of ligand binding to the heme, these contacts are altered (Figure 15.31). Hemoglobin, as a conformationally dynamic molecule, consists of two dimeric halves, an a1/1-subunit pair and an a2/2-subunit pair. Each a/3 dimer moves as a rigid body, and the two halves of the molecule slide past each other upon oxygenation of the heme. The two halves rotate some 15° about an imaginary pivot passing through the a//-subunits; some atoms at the interface between a/ dimers are relocated by as much as 0.6 nm.

Movement of the Heme Iron by Less Than 0.04 nm Induces the Conformational Change in Hemoglobin

In deoxyhemoglobin, histidine F8 is liganded to the heme iron ion, but steric constraints force the Fe2+:His-N bond to be tilted about 8° from the perpendicular to the plane of the heme. Steric repulsion between histidine F8 and the nitrogen atoms of the porphyrin ring system, combined with electrostatic repulsions between the electrons of Fe2+ and the porphyrin ^-electrons, forces the iron atom to lie out of the porphyrin plane by about 0.06 nm. Changes in electronic and steric factors upon heme oxygenation allow the Fe2+ atom to move about 0.039 nm closer to the plane of the porphyrin, so now it is displaced only 0.021 nm above the plane. It is as if the O2 were drawing the heme Fe2+ into the porphyrin plane (Figure 15.32). This modest displacement of 0.039 nm seems a trivial distance, but its biological consequences are far-reaching. As the iron atom moves, it drags histidine F8 along with it, causing helix

FIGURE 15.30 • Side view of one of the two a/ dimers in Hb, with packing contacts indicated in blue. The sliding contacts made with the other dimer are shown in yellow. The changes in these sliding contacts are shown in Figure 15.31. (Irving Geis)

FIGURE 15.31 • Subunit motion in hemoglobin when the molecule goes from the (a)

deoxy to the (b) oxy form. (Irving Geis)

FIGURE 15.31 • Subunit motion in hemoglobin when the molecule goes from the (a)

deoxy to the (b) oxy form. (Irving Geis)

F, the EF corner, and the FG corner to follow. These shifts are transmitted to the subunit interfaces, where they trigger conformational readjustments that lead to the rupture of interchain salt links.

The Oxy and Deoxy Forms of Hemoglobin Represent Two Different Conformational States

Hemoglobin resists oxygenation (see Figure 15.22) because the deoxy form is stabilized by specific hydrogen bonds and salt bridges (ion-pair bonds). All of these interactions are broken in oxyhemoglobin, as the molecule stabilizes into a new conformation. A crucial H bond in this transition involves a particular tyrosine residue. Both a- and ¡-subunits have Tyr as the penultimate C-termi-nal residue (Tyr a140 = Tyr HC2; Tyr ¡145 = Tyr HC2, respectively2). The phenolic —OH groups of these Tyr residues form intrachain H bonds to the peptide C=O function contributed by Val FG5 in deoxyhemoglobin. (Val FG5 is a93 and ¡98, respectively.) The shift in helix F upon oxygenation leads to rupture of this Tyr HC2:Val FG5 hydrogen bond. Further, eight salt bridges linking the polypeptide chains are broken as hemoglobin goes from the deoxy to the oxy form (Figure 15.33). Six of these salt links are between different subunits. Four of these six involve either carboxyl-terminal or amino-terminal amino acids in the chains; two are between the amino termini and the carboxyl termini of the a-chains, and two join the carboxyl termini of the ¡-chains to the e-NH3+ groups of the two Lys a140 residues. The other two interchain electrostatic bonds link Arg and Asp residues in the two a-chains. In addition, ionic interactions between Asp ¡94 and His ¡146 form an intrachain salt bridge in each ¡-subunit. In deoxyhemoglobin, with all of these interactions intact, the C-termini of the four subunits are restrained, and this conformational state is termed T, the tense or taut form. In oxyhemoglobin, these C-termini have almost complete freedom of rotation, and the molecule is now in its R, or relaxed, form.

2C here designates the C-terminus; the H helix is C-terminal in these polypeptides. "C2" symbolizes the next-to-last residue.

FIGURE 15.33 • Salt bridges between different subunits in hemoglobin. These noncova-lent, electrostatic interactions are disrupted upon oxygenation. Arga and His^ are the C-termini of the a- and ^-polypeptide chains. (a) The various intrachain and interchain salt links formed among the a- and ^-chains of deoxyhemoglobin. (b) A focus on those salt bridges and hydrogen bonds involving interactions between N-terminal and C-terminal residues in the a-chains. Note the Cl ion, which bridges ionic interactions between the N-terminus of and the R group of Arga . (c) A focus on the salt bridges and hydrogen bonds in which the residues located at the C-termini of ^-chains are involved. All of these links are abolished in the deoxy to oxy transition. (Irving Geis)


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