The crystal structure of a tetrameric hemoglobin in a partial hemichrome state

Abstract

Tetrameric hemoglobins are the most widely used systems in studying protein cooperativity. Allosteric effects in hemoglobins arise from the switch between a relaxed (R) state and a tense (T) state occurring upon oxygen release. Here we report the 2.0-A crystal structure of the main hemoglobin component of the Antarctic fish Trematomus newnesi, in a partial hemichrome form. The two alpha-subunit iron atoms are bound to a CO molecule, whereas in the beta subunits the distal histidine residue is the sixth ligand of the heme iron. This structure, a tetrameric hemoglobin in the hemichrome state, demonstrates that the iron coordination by the distal histidine, usually associated with denaturing states, may be tolerated in a native-like hemoglobin structure. In addition, several features of the tertiary and quaternary organization of this structure are intermediate between the R and T states and agree well with the R --> T transition state properties obtained by spectroscopic and kinetic techniques. The analysis of this structure provides a detailed pathway of heme-heme communication and it indicates that the plasticity of the beta heme pocket plays a role in the R --> T transition of tetrameric hemoglobins.

Figure 1

Electron density maps (contoured at 2.5 σ) corresponding to the α (A) and β (B) heme regions.

Figure 2

Stereo drawing showing the scissors-like motion of the β chain EF corner occurring upon hemichrome formation. The EF regions of HbTn[α(CO)β(hemi)]₂ (cyan) and HbTnCO (red) are shown after superimposition of the F helix. For clarity the propionate groups of heme have been omitted.

Figure 3

Distribution of the C^α–C^α distances between the proximal and the distal histidine residues in 276 α and β chains belonging to 80 tetrameric Hbs reported in the PDB (38). The value corresponding to the β chain of HbTn[α(CO)β(hemi)]₂ is colored in black.

Figure 4

Stereo drawing showing the relative orientation of His-97β side chain and helix C of the α chain at the α₁β₂ of liganded and unliganded Hbs. HbTnCO, HbTn[α(CO)β(hemi)]₂, HbTb-deoxy and the R2 carbonmonoxy form of human Hb are colored in red, cyan, green, and yellow, respectively. The residues of helix C of the α chains have been used in the superimposition. For clarity only helix C of HbTn[α(CO)β(hemi)]₂ is shown.

Figure 5

Structural modifications of the α chain produced by hemichrome formation in the β chain. HbTn[α(CO)β(hemi)]₂ and HbTnCO are colored in gray and black, respectively.

Figure 6

C^α trace of HbTn[α(CO)β(hemi)]₂ (cyan), HbTbCO (purple), and HbTb-deoxy (green). The residues of the α₁β₁ dimers have been superimposed. Residues belonging to the α₁β₁ and α₂β₂ dimers are mostly located in the lower and in the upper part of the figure, respectively. The labeled residues Asp-5, together with Lys-95 and Pro-47, belong to β₂ and α₂ chains, respectively. The axis required to superimpose the α₂β₂ dimers of HbTbCO and HbTb-deoxy is normal to the figure and its position is marked by a black circle. The gray circle represents the intersection of the axis required to superimpose the α₂β₂ dimers of HbTn[α(CO)β(hemi)]₂ and HbTb-deoxy with a plane parallel to the figure and passing through the center of mass of the tetramer. The two axes form an angle of 15°.