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

In order to detect and model the effect of functional chain heterogeneity o n Nernst plots for heme proteins, we examined the redox properties of vario us myoglobins (Mbs) and their mixtures using an improved spectroelectrochem ical method. Specific redox responses and formal half potentials (E-1/2) we re obtained for Aplysia, horse, and sperm whale Mbs, as well as 1:1 mixture s of Mba consisting of Aplysia/sperm whale, sperm whale/horse, and horse/Ap lysia. Linear Nernst plots with slopes near unity were observed for horse, sperm whale, and Aplysia Mbs, with E-1/2 values of 14, 19, and 96 mV (vs. N HE) respectively, consistent with previous reports using indirect methods. The Nernst plot responses for mixtures of some of these Mbs allowed us to e valuate and model the non-Nernstian behavior that results from intrinsicall y different values of E-1/2 and from incomplete spectral overlap. The data demonstrate that increasing the E-1/2 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 (n (Nerns)t). Comparison of Nernst plots for redox data with Hill plots for O- 2 binding data shows that the redox process is more affected by the structu ral differences in the distal heme pockets of the Mbs studied than is O-2 b inding. Similar effects of chain heterogeneity may give rise to disproporti onate reductions in the slopes of Nernst and Hill plots for hemoglobins (Hb s). This possibility is discussed in relation to Hbs investigated for redox and O-2 binding activity in our laboratories where we find n(Nernst) to be commonly less than n(Hill) over a range of experimental conditions. (C) 19 99 Elsevier Science S.A. All rights reserved.