Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins

Abstract

Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.

Figure 1

Deconvolution of sequence ambiguity from mass spectra using X-ray crystallography. (a) Mass spectrum for the N-terminal peptide fragment of snow leopard Hb β chain. The top line reading from right to left is the sequence. The identity of the third residue is ambiguous. (b) Electron density (contoured at 1.5σ) of the β-chain N-terminus. The density for the third residue indicates that Leu is the appropriate choice.

Figure 2

Contrasting neutron density (contoured at 1σ) near the switch and hinge regions of T-state and R-state hemoglobin. (a) T-state human deoxy­hemoglobin; (b) R-state equine cyanomethemoglobin.

Figure 3

Ribbon backbone models for two different B-state hemoglobins: (a) snow leopard CNmetHb, (b) human recombinant 1.1 CNmetHb.

Figure 4

Ribbon backbone models of R-state and T-state hemoglobin. Note the larger central opening for the T state, which is comparable to the larger opening seen in the B state (Fig. 3 ▶). (a) Equine CNmetHb, (b) human deoxyhemoglobin.