Faulkner K M, Bonaventura C, Crumbliss A L
Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA.
J Biol Chem. 1995 Jun 9;270(23):13604-12. doi: 10.1074/jbc.270.23.13604.
Spectroelectrochemical techniques are described which enable us to compare anion effects on redox curves of structurally distinct hemoglobins with oxygenation curves obtained under equivalent conditions. Nernst plots for tetrameric vertebrate Hbs show evidence of cooperativity, with the T state conformation more resistant to oxidation than the R state. Anions shift the conformation toward the T state and decrease the ease of oxidation, with variations in anion sensitivity similar to those observed in oxygen equilibria. Oxygen binding, unlike electron exchange, is known to be subject to steric constraints that vary considerably in natural and engineered hemoglobins that have differences in the distal residues of the heme pocket. Since oxidation curves are not subject to steric hindrance, anion-induced differences between the oxidation and oxygenation curves can be indicative of anion-induced alterations in the stereochemistry of the heme pocket that alters the ease of ligand entry or exit. Addition of inositol hexaphosphate to solutions of Hb A in 0.2 M nitrate generates such differences: the ease of electron abstraction from deoxy (T state) Hb A is unaffected, while, as previously reported, the oxygenation of deoxy (T state) Hb A is greatly hindered. The difference between inositol hexaphosphate effects on initial stages of oxidation and oxygenation indicates that the explanation for "multiple T states" in oxygen binding lies in the ability of the polyanion to greatly increase steric hindrance to ligand entry, without appreciable changes in the electronic features of the heme environment.
