title: Solution NMR study of the structural basis of the Bohr effect in the monomeric hemoglobins from Chironomus thummi thummi.
abstract: The larva of the midge Chironomus thummi thummi possesses two monomeric hemoglobins. HbIII and HbIV, with extensive sequence homology, which exhibit marked but differential Bohr effects (pH influence on ligand affinity). These Hbs serve as ideal models for allosteric control of ligand affinity via tertiary-only structural changes. The cyanomet derivatives of these two Hbs have been shown to possess essentially indistinguishable heme cavity structures in solution at low pH (Zhang et al., 1996) that are also very similar to that of the low pH form of HbIII in the crystal (Steigemann & Weber, 1979). 2D 1H NMR has been utilized to elucidate the solution heme cavity structure of the alkaline form of the cyanomet derivatives of HbIII and HbIV to identify the Bohr proton binding site and characterize the nature of the structural changes that accompany the allosteric transition. Significant structural changes with pH have been identified in two regions of the heme cavity, near the axial His and at the junction of pyrroles B and C. The Bohr proton site is identified as His94, which at low pH makes a salt bridge to the terminal Met136. The rupture of this salt bridge at high pH leads to the expulsion of the Met136 side chain next to the His F8 ring where it serves as a spacer between the heme and F-helix, and leads to a cascade of side chain reorientations in the densely packed hydrophobic interior involving five Phe (65, 66, 128, 129, 133), Val132, and Ile69, all on the E- and H-helices. The terminal member of the cascade, Phe65, which acts as a spacer between the E- and F-helices at low pH, is rotated toward the heme plane. The conversion of the low pH, low-affinity "tense" to the high pH, high-affinity "relaxed" state is primarily due to the removal of the Met136 and Phe65 spacers. A central residue in transmitting the Bohr effect from His94 to Phe65 is residue 132. In HbIV, Val132 provides a cavity in the hydrophobic core to readily accommodate the initial step in rotating the Phe129 side chain. In HbIII, the Ile132 provides tight packing to all neighboring side chains and hence would inhibit the rotation of the Phe129 side chain. It is proposed that the lone internal residue difference between HbIII (Ile132) and HbIV (Val132) is the primary basis for the different amplitudes of their Bohr effect.
authors.affiliation: Department of Chemistry, University of California, Davis 95616, USA.
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Gersonde
authors.forename: G N
authors.initials: GN
authors.lastname: La Mar
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D002683:Chironomidae / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011522:Protons; D012996:Solutions; D013329:Structure-Activity Relationship; D017186:Titrimetry
chemical_list: D006454:Hemoglobins; D011522:Protons; D012996:Solutions; D006418:Heme