Oxidative titrations of reduced cytochrome aa3: anisotropic extinction behavior observed in the heme alpha-band region.

Abstract

Direct chemical titrations of reduced, purified cytochrome aa3 were monitored at 604 nm. Anaerobic oxidation by potassium ferricyanide or 1,1'-bis(hydroxymethyl)-ferricinium ion was compared with the natural substrate, molecular oxygen. The four electrons from reduced cytochrome aa3 were donated to one oxygen molecule, resulting in a linear titration with only fully oxidized or fully reduced enzyme molecules present. Unlike the linear or sigmoidal titrations which resulted from various reductive titrations reported previously, pronounced hyperbolic curves were obtained with the chemical oxidants. The midpoint potential values exhibited by the four metal centers of cytochrome aa3 were determined by computer simulation of the titration curves. A high potential pair of components (heme a = 340 mV, Cu = 340 mV) and a low potential pair (heme a = 220 mV, Cu = 240 mV) were observed, consistent with literature values. Unique to these equilibrium studies was a split in the heme a extinction coefficients at 604 nm. The low potential heme a component contributed 80-85% to the total absorbance change. A polarographic assay was used to verify that the integrity of cytochrome aa3 remained intact during equilibrium titrations spanning several hours. These experimental results indicate that simple chemical reversibility does not exist for cytochrome aa3 under anaerobic conditions.