Conformational fluctuations in deoxy hemoglobin revealed as a major contributor to anionic modulation of function through studies of the oxygenation and oxidation of hemoglobins A0 and Deer Lodge beta2(NA2)His --> Arg.
Organisms rely on regulation at the molecular level, such as the allosteric regulation of hemoglobin (Hb) function by anions, to meet challenges presented by changing environmental and physiological conditions. A comparison of the effects of anions on oxygenation, oxidation, and sulfhydryl reactivity of Hb leads us to suggest that a large and significant part of the shift in oxygen affinity brought about by anion binding occurs as a result of increased conformational rigidity of the T state of deoxy Hb. As conformational rigidity increases, it becomes increasingly difficult for subunits in the deoxygenated T-state tetramer to assume higher oxygen affinity forms (T', T", T"'...) with less steric hindrance. The oxygen affinity reflects the average of the rapidly equilibrating conformations within the T state and is correspondingly decreased when anion levels are increased. The initial stage of the oxidation of Hb is relatively insensitive to steric alterations and thus reflects, primarily, the electronic aspects of the quaternary (T, T', T", T"'...) <--> equilibrium. We show that the reactivity of the sterically obscured sulfhydryl of beta93 Cys in deoxy Hb is much greater in chloride-free buffers than in buffers with added chloride. Anion-induced decreases in the extent and frequency of conformational fluctuations of subunits within the T-quaternary state thus reduce sulfhydryl reactivity as well as oxygen affinity. This parallel in anionic control of function allowed us to test, and disprove, the possibility that uncompensated positive charges in the central cavity of Hb Deer Lodge increase the frequency and extent of conformational fluctuations in its deoxy structure. This Hb variant exhibits increased anion sensitivity, increased oxygen affinity, and increased ease of oxidation, but without increased reactivity of its sulfhydryl groups, indicating that active-site alterations in deoxy Hb Deer Lodge are primarily electronic and not associated with increased conformational fluctuations within its T state. The restoration of normal properties in Hb Deer Lodge by addition of anions reinforces our conclusion that anionic control can be exerted through both steric and electronic alterations. The anionic control of fluctuations within the T state of Hb illustrates an important principle of macromolecular structure-function relationships: that functional regulation can be achieved by alterations in conformational rigidity.
Bonaventura
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Duke University Marine Laboratory, Beaufort, North Carolina 28516, USA.
Tesh
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Faulkner
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Kraiter
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Crumbliss
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D000838:Anions / Q000494:pharmacology; D001665:Binding Sites; D002712:Chlorides / Q000494:pharmacology; D006441:Hemoglobin A / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006455:Hemoglobins, Abnormal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D013439:Sulfhydryl Reagents; C036096:hemoglobin Deer Lodge; C028881:hemoglobin A(0); C100003:deoxyhemoglobin; D006441:Hemoglobin A; D010100:Oxygen