Spectroelectrochemistry of heme proteins: effects of active-site heterogeneity on Nernst plots.

In order to detect and model the effect of functional chain heterogeneity on Nernst plots for heme proteins, we examined the redox properties of various myoglobins (Mbs) and their mixtures using an improved spectroelectrochemical method. Specific redox responses and formal half potentials (E1/2) were obtained for Aplysia, horse, and sperm whale Mbs, as well as 1:1 mixtures of Mbs consisting of Aplysia/sperm whale, sperm whale/horse, and horse/Aplysia. Linear Nernst plots with slopes near unity were observed for horse, sperm whale, and Aplysia Mbs, with E1/2 values of 14, 19, and 96 mV (vs. NHE) respectively, consistent with previous reports using indirect methods. The Nernst plot responses for mixtures of some of these Mbs allowed us to evaluate and model the non-Nernstian behavior that results from intrinsically different values of E1/2 and from incomplete spectral overlap. The data demonstrate that increasing the E1/2 differences between the components of a Mb mixture increases the changes in shape of the resulting Nernst plots, the dominant effect being a decrease in the observed Nernst coefficient (nNernst). Comparison of Nernst plots for redox data with Hill plots for O2 binding data shows that the redox process is more affected by the structural differences in the distal heme pockets of the Mbs studied than is O2 binding. Similar effects of chain heterogeneity may give rise to disproportionate reductions in the slopes of Nernst and Hill plots for hemoglobins (Hbs). This possibility is discussed in relation to Hbs investigated for redox and O2 binding activity in our laboratories where we find nNernst to be commonly less than nHill over a range of experimental conditions.