Hemoglobin (haemoglobin, Hb, Hgb) red cells in blood carry O 2 from lung to tissues by hemoglobin, a 4-subunit protein having an O 2 -binding prosthetic group, heme, that gives blood its color (Hb also carries (some) CO 2 and H + back to the lung) one of the first proteins the structure of which has been determined for (Perutz, 1959; Noble Prize in Chemistry shared with Kendrew in 1962) contrary to Hb, myoglobin (Mb) is a monomeric O 2 -storage molecule, evolutionary related to Hb, a very similar structure to Hb (globin fold) Hb can use 90% of its full O 2 -binding potential, while Mb can use only 7% in Hb there is a so-called cooperativity, so O 2 -binding to one chain potentiates O 2 -binding to the rest of the 3 chains (same true for release) H + - and CO 2 -binding to Hb also modulate O 2 -binding the hypothesis that alteration in amino acid sequence may lead to disease was first proven with Hb and Mb (sickle-cell anemia; single mutation in Hb)

O 2 -free or O 2 -bound states exist: deoxy-Hb/Mb or oxy-Hb/Mb high  -helical content connected by  -turns in Hb/Mb with one O 2 -binding site/monomer on each heme prosthetic group under normal conditions, heme binds Fe 2+, there are a 5 th and a 6 th coordi- nation site for Fe on each side of the heme plane the 5 th coordination of Fe takes place with a His side-schain (imidazole) of the protein (proximal His); the 6 th site is unoccupied in the deoxy-pro- tein (ready for binding O 2 ) Fe is too big for the hole in the middle of the porphyrin ring (deoxy-Mb/Hb)

upon oxygenation: rearranged electon-structure of Fe upon O 2 -binding results in a slightly smaller size that now fits in the middle of the tetrapyrrole ring (magnetic properties also change for Hb and that is the basis of functional magnetic resonance imaging, fMRI, one of the most powerful methods of examining brain function) Linus Pauling predicted this as well in 1936, 25 years ahead of time

O 2 -bound Fe acts rather as a Fe 3+ - O 2 - (superoxide anion) complex (charge- transfer complex, mixture of resonance structures); it is crucial to release oxygen as O 2 because superoxide is a reactive oxygen species (ROS) and can generate further harmful species that damage various cellular compo- nents (proteins, DNA, lipids, membranes, etc.) and this would leave Fe as Fe 3+ that with heme would constitute for metmyoglobin/methemoglobin that does not bind O 2 (O 2 -storage capacity is lost)…structural features of Mb/Hb stabilize the O 2 -Hb/Mb complex in such a way that will assure that oxygen will be released as O 2 there is another His (distal His) that donates a H-bond to the bound O 2 (the superoxide character of bound O 2 strengthens this interaction; the protein part of Hb/Mb controls O 2 -binding and release) Mb’s P 1/2 value (50% of saturation of available binding sites) for O 2 is at 2 torr (Hg mm), simple saturation curve

Hb`s O 2 -binding curve looks like an “S” letter called a sigmoid curve (signi- ficantly weaker O 2 -binding than for Mb at the same O 2 -tension, P 1/2 =26 torr; this is true in red blood cells where Hb binds a special molecule, 2,3-bis- phosphoglycerate, as well, that lowers Hb`s affinity to O 2 significantly)

Physiological importance of sigmoidal O 2 -binding in the lungs partial pressure of O 2 is high (100 torr, 98% of Hb binds O 2 ), in actively metabolizing tissues it is 20 torr (32% saturation; in resting state 40 torr); 98-32=66% of the binding sites contribute to O 2 -transport (these numbers would be 98% vs. 91% for Mb, only 7% difference – too small window to work with, Mb binds O 2 too tight for O 2 -transport) these numbers would be for a protein with theoretically optimal affinity for O 2, 63% and 25% (38% difference), cooperativity is the best solution for delivering O 2 to tissues (~10x better than it would be possible with Mb)

100 torr (lung) to 40 torr (resting muscle, 60 torr drop in partial pressure) means 98% to 77%=21% drop in O 2 -saturation of Hb 40 torr (resting muscle) to 20 torr (in exercise, 20 torr drop) means 77% to 32%=45% drop in O 2 -binding of Hb steepest part of the O 2 -binding curve is right there where the most O 2 is needed, so when we switch from resting to exercise

How is cooperativity delivered at the atomic level? O 2 -binding sites are far away from each other, direct interaction is not possible upon O 2 -binding the  1-  1 and  2-  2 dimers rotate ~15 o with respect to one another (substantial conformational change, the connecting interface changes the most, the dimers themselves not that much, the dimers are freer to move and O 2 -binding sites are free of strain and are capable of binding O 2 with a much higher affinity in the oxygenated state) Tense state, deoxy-Hb, constrained by subunit- subunit interactions Relaxed state, fully oxygenated form

Concerted or MWC model: only two states exist, T and R states, in the R state O 2 -binding is much stronger, occupying more and more binding sites with O 2 shifts the equi- librium towards the R state Sequential model: binding of a ligand changes the conformation of the subunit that it binds to, which consequently will change the conformation of a neighboring subunit so that its affinity for O 2 would be increased and so on neither model is fully describing the actual mechanism of action of Hb

How does O 2 -binding at one site shift the T to R equilibrium for the tetramer? upon O 2 -binding Fe moves inplane which pulls also on proximal His proximal His is part of an  -helix that also moves with the His the C-terminus of this helix lies in the interface of the two  dimers and the movement of this C-terminal region triggers the T-to-R transformation of Hb the structural transition at the iron ion in one of the subunits is directly transmitted to the other subunits (the rearrangement of the inter-dimer interface makes communication between subunits possible enabling the cooperative binding of O 2

2,3-BPG and the O 2 -affinity of Hb pure Hb binds O 2 much tighter than Hb in red cells and the reason is the presence of 2,3-BPG in red cells (~2 mM, just as much as Hb itself) without 2,3-BPG binding to Hb, Hb would be able to release only 8% of its O 2 -load in tissues crystal structure of deoxy-Hb bound to 2,3-BPG reveals that a single mole- cule of 2,3-BPG binds to a Hb tetramer in a pocket in the center of the tetramer; this pocket exists only in the T-form and gets collapsed in the R- form (and 2,3-BPG gets released) in order to make the T-to-R transition happen 2,3-BPG must dissociate off of Hb in the presence of 2,3-BPG more O 2 -binding sites must be occupied to induce the T-to-R transition, hence Hb stays in the lower affinity T state until higher O 2 concentrations are available 2,3-BPG is an allosteric effector (see later)

Fetal Hb fetal Hb consists of 2  and 2  chains (latter has 72% sequence homo- logy to the  Hb chain) H143S is an important change (mutation) as H143 is part of the 2,3-BPG binding site; this mutation removes positive charges from the binding pocket lowering the affinity of 2,3-BPG to this site consequently fetal Hb will bind O 2 with higher affinity than maternal Hb elegant solution from nature to solve an important biological problem (to transport O 2 from mother to fetus)

The Bohr effect rapidly metabolizing tissues such as contracting muscle generate a lot of H + and CO 2 Hb is able to release more O 2 where need augments (e.g. in contracting muscle) by the action of H + and CO 2 (also allosteric effectors; Bohr effect) lowering pH lowers O 2 -affinity of Hb (from 7.4 (lung) to 7.2 (tissue) with an 80 torr drop in pressure this amounts to a 77% (rather than 66% at pH=7.4) total carrying capacity) the mechanism of action is revealed, but not discussed here (rather complex mechanism) CO 2 action is also partly due to acidification (carbonic acid for- mation)

reaction with N-terminus (switch of charge) carbamate termini participate in salt-bridge interactions in the  inter-dimer interface and stabilize the T-state (lower O 2 -affinity, also 14% of CO 2 -transport)

Sickle-Cell Anemia (SCA) large fibrous aggregates of Hb, distorted red cells that clog small capilla- ries and impair normal blood flow (symptoms: painful swelling of extremities, higher risk for stroke, anemia; 1% of West Africans suffer from this dise- ase) E6V mutation is the responsible in the  chain, the mutant Hb is called HbS, deoxy-HbS has a very low solubility, both alleles are affected in disease Why Africa? people heterozygous for SCA are resistant to malaria