Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin

Abstract

The dynamic and functional properties of mutant deoxyhemoglobins in which either the ␤-globin Val 67 (E11) or the ␣-globin Val 62 (E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified ␣ chain and ␤ chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the ␣ chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the ␤ chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified ␣ chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the ␣ chain distal heme pocket can bridge by H bonding between Thr 62 (E11) and His 58 (E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified ␤ chains is not observed by x-ray crystallography.

Properties of Human Hemoglobins with Increased Polarity in the α- or β-Heme Pocket

“…As previously discussed, the Val 3 Thr substitution does not modify the steric characteristics of the heme pocket (7,10,11); therefore, this system represents an excellent model to investigate the effect of modified heme pocket polarity on the ligand binding parameters and their correlation with IR spectroscopy. For this analysis, the kinetic curves have been analyzed in terms of two exponentials of equal amplitudes (describing the contributions from ␣-and ␤-subunits).…”

“…From Fig. 3, it can be seen that, at variance with the deoxy derivatives (10), in the carbonmonoxy derivatives, a remarkable effect (red shift) was observed for the ␤V(E11)T mutant, whereas the recombinant wild-type proteins and the ␣V(E11)T mutant exhibited almost the same behavior as HbA. This shows that, for the CO derivatives, the Val(E11)T substitution in the ␤-chain considerably alters the local electric field at the chromophore (and therefore the pure electronic transi- Increased ␣/␤-Heme Pocket Polarity Dynamic/Functional Effectstion frequency 0 ), whereas the effect is smaller for the ␣-chain mutation.…”

“…Proteins and experimental methods were nearly the same as those described by Cupane et al (10). The following differences are noted.…”

“…Contrary to the deoxy derivatives, in which the equilibrium position of the iron atom out of the mean heme plane gives rise to a nonsymmetric inhomogeneous broadening of the Soret band (see Cupane et al (10) for an in-depth discussion), in the CO derivatives, the effect of conformational heterogeneity can be modeled as a gaussian distribution of 0 -0 transition frequencies and gives rise to the additional term in 2 in Equation 4. IR Spectra-Infrared spectra were recorded at room temperature using a Perkin-Elmer 1600 series FT-IR spectrometer.…”

The Oxygen Transport System in Three Species of the Boreal Fish Family Gadidae

“…In deoxy-HbA, Val ␤E11 overlaps the ligand-binding site and is considered to play a key role in controlling the oxygen affinity (40). It has also been shown that the replacement of Val ␤E11 with Ile affects the kinetic parameters of oxygen binding (41), because of the larger sec-butyl group that sterically hinders access of ligands to the heme iron. The contact surface area between the ␤-heme and Ile(E11) is 59 Å 2 in T. newnesi Hb C (displaying the substitution Val ␤E11 3 Ile ␤E11 at the distal side of the heme pocket), whereas it is 50 Å 2 in Trematomus bernacchii Hb, which has Val (42).…”