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

Abstract

Significance: Over the past 50 years, the mechanisms for O 2 storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and timescales from seconds to picoseconds. Recent Advances: In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O 2 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 O 2 , which selectively enhances O 2 affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O 2 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. Critical Issues: These conclusions indicate that there are often compromises between O 2 transport function, holoprotein stability, and the efficiency of assembly. Future Directions: 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.

Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli

“…Extensive studies of myoglobin have shown that during expression, the stability of the apoglobin is imperative to achieve high yields ( Samuel et al, 2015 ). In the case of Hb, this dependence on the stability of the apo-subunits on yields has not yet been fully confirmed, probably due to the additional subunit association factors complicating the Hb folding/precipitation processes ( Olson 2020 ).…”

“…Examples of mutations that have been shown to increase yield in recombinant fermentation of HbA is the β82 (EF6) Lys→Asp (Hb Providence asp ( Moo-Penn et al, 1976 )) ( Weickert et al, 1999 ), α15 (A13) Gly→Ala, β16 (A13) Gly→Ala, and β116 (G18) His→Ile ( Graves et al, 2008 ). For an overview of mutational strategies, Olson recently summarized 50 years of research and mutational work on myoglobin and Hb and highlights the importance of polypeptide chain stability at different stages of globin assembly ( Olson 2020 ). However, the impact of net charge density of the surface of Hb regarding yields in recombinant expression has not been the subject of any comprehensive studies to the best of our knowledge.…”

Allosteric control of hemoglobin S fiber formation by oxygen and its relation to the pathophysiology of sickle cell disease

“…Oxygen binding is close to equilibrium as red cells pass through the tissues because the dissociation, binding (90), and conformation rates (44) are comparable to or faster than the transit time from the arteries to the venules. As a result, the oxygen binding measurements for normal hemoglobin measured Comparison of average measured and theoretically calculated fraction SS cells sickled vs. time for slow deoxygenation to a final gas mixture of 95% nitrogen and 5% oxygen.…”

“…Oxygen binding is close to equilibrium as red cells pass through the tissues because the dissociation, binding ( 90 ), and conformation rates ( 44 ) are comparable to or faster than the transit time from the arteries to the venules. As a result, the oxygen binding measurements for normal hemoglobin measured on the tens of minutes time scale that began over 100 y ago ( 91 ) have physiological significance even though deoxygenation and reoxygenation in vivo occurs much faster.…”

Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

“…Globins contain redox active heme centers with either 5- or 6-coordinate iron and, in the presence of O 2 , degrade NO by NO dioxygenation (NOD) with the resulting formation of NO 3 − ( 5 , 7 , 14 , 15 , 16 , 17 ). The efficiency of NOD activity of various mammalian globins, hemoglobin (Hb), myoglobin (Mb), cytoglobin (Cygb), and neuroglobin (Ngb) varies based on differences in their heme center and overall structure.…”