Site-Directed Mutagenesis in Hemoglobin: Functional and Structural Role of the Penultimate Tyrosine in the .alpha. Subunit

Abstract

The penultimate tyrosine in the hemoglobin subunit is considered to be one of the most important residues for the normal structure and function of hemoglobin. To elucidate the functional and structural role of the penultimate residue in the alpha-subunit, we prepared new artificial mutants; Hb Y140 alpha Q, in which Tyr-140 alpha is replaced by a nonaromatic residue, Gln, and Hb Y140 alpha F, which loses its hydrogen bond to Val-93 alpha by the substitution of Phe for Tyr. HB Y140 alpha Q exhibited a markedly increased oxygen affinity and almost completely diminished cooperativity, whereas Hb Y140 alpha F showed similar but less extensively impaired function, indicating that the aromatic residue at the penultimate position in the alpha-subunit contributes to the stabilization of the T-quaternary structure as does the corresponding residue in the beta-subunit. However, the deoxygenated forms of these mutants bear significant T-state character in their spectroscopic properties observed at high protein concentrations. The tetramer-dimer equilibrium data of the mutants suggested that a significant part of the functional alterations observed for dilute solution appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated form. Therefore, we can conclude that the penultimate tyrosine in the alpha-chain plays a key role not only in the stabilization of the T-state but also in the subunit assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and Functional Effects of Pseudo-module Substitution in Hemoglobin Subunits

“…Fig. 5A illustrates gel chromatogram of the carbonmonoxy chimeric globins in the presence and absence of the native subunits, and the centroid elution volumes of the samples as a function of protein concentration (16,17) are shown in Fig. 5B.…”

“…The buffer used for the chromatography was 50 mM Tris, in the presence of 0.1 M NaCl, and 1 mM Na 2 EDTA, pH 7.4, and the flow rate was 7 ml/h. The eluted fractions were monitored by absorption at the Soret band (16,17). Dimer-tetramer dissociation constant of the samples was determined by concentration dependence of the centroid elution volume over the range from 0.5 to 200 M (16,17).…”

“…The eluted fractions were monitored by absorption at the Soret band (16,17). Dimer-tetramer dissociation constant of the samples was determined by concentration dependence of the centroid elution volume over the range from 0.5 to 200 M (16,17). The following functional dependence of the elution volume (V e ) versus protein concentration (C T ) allows 1 Wild-type subunit represents the protein expressed in Escherichia coli, and a methionine residue is located at the N-terminal.…”

“…The NMR spectrum in the hyperfine-shifted region at 12-28 ppm is also suggestive of absence of the specific interactions between these two subunits (36). On the basis of x-ray structural studies, two sliding contact regions, C2-CD1 and FG3-G7, are essential for the allostericity in hemoglobin A (16,(42)(43)(44)(45)(46)(47)(48). However, the C2-CD1 region of the ␤␣(PM3)-subunit is derived from the ␤-subunit (Fig.…”

Substitution of the Heme Binding Module in Hemoglobin α- and β-Subunits

“…The eluted fractions were monitored by absorption at the Soret band (420 nm). Tetramer-dimer dissociation constants of the samples were determined by the concentration dependence of the elution volume in the column over the range from 0.5 to 800 M (23,24). The dependence of the centroid elution volume V e versus protein concentration (C T ), Equation 1, allows us to determine the dimer-tetramer equilibrium constants for the samples,…”