Lessons Learned from 50 Years of Hemoglobin Research: Unstirred and Cell-Free Layers, Electrostatics, Baseball...

Over the past 50 years, the mechanisms for O₂ storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and timescales from seconds to picoseconds. In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O₂ uptake by mammalian red cells is limited by diffusion through unstirred water layers adjacent to the cell surface and across cell-free layers adjacent to vessel walls. (ii) In most vertebrates, hemoglobins (Hbs) and myoglobins (Mbs), the distal histidine at the E7 helical position donates a strong hydrogen bond to bound O₂, which selectively enhances O₂ affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O₂ binding to mammalian Hbs and Mbs occurs by migration of the ligand through a channel created by upward rotation of the His(E7) side chain, capture in the empty space of the distal pocket, and then coordination with the ferroprotoporphyrin IX (heme) iron atom. (iv) The assembly of Mbs and Hbs occurs by formation of molten globule intermediates, in which the N- and C-terminal helices have almost fully formed secondary structures, but the heme pockets are disordered and followed by high-affinity binding of heme. These conclusions indicate that there are often compromises between O₂ transport function, holoprotein stability, and the efficiency of assembly. However, the biochemical mechanisms underlying these conclusions provide the framework for understanding globin evolution in greater detail and for engineering more efficient and stable globins.