A SPECTROELECTROCHEMICAL METHOD FOR DIFFERENTIATION OF STERIC AND ELECTRONIC EFFECTS IN HEMOGLOBINS AND MYOGLOBINS

Spectroelectrochemical techniques are described which enable us to com pare anion effects on redox curves of structurally distinct hemoglobin s with oxygenation curves obtained under equivalent conditions. Nernst plots for tetrameric vertebrate Hbs show evidence of cooperativity, w ith the T state conformation more resistant to oxidation than the R st ate. Anions shift the conformation toward the T state and decrease the ease of oxidation, with variations in anion sensitivity similar to th ose observed in oxygen equilibria. Oxygen binding, unlike electron exc hange, is known to be subject to steric constraints that vary consider ably in natural and engineered hemoglobins that have differences in th e distal residues of the heme pocket. Since oxidation curves are not s ubject to steric hindrance, anion-induced differences between the oxid ation and oxygenation curves can be indicative of anion-induced altera tions in the stereochemistry of the heme pocket that alters the ease o f ligand entry or exit. Addition of inositol hexaphosphate to solution s of Hb A in 0.2 M nitrate generates such differences: the ease of ele ctron abstraction from deoxy (T state) Hb A is unaffected, while, as p reviously reported, the oxygenation of deoxy (T state) Hb A is greatly hindered. The difference between inositol hexaphosphate effects on in itial stages of oxidation and oxygenation indicates that the explanati on for ''multiple T states'' in oxygen binding lies in the ability of the polyanion to greatly increase steric hindrance to ligand entry, wi thout appreciable changes in the electronic features of the heme envir onment.