Potentiometric Analysis of the Purified Cytochrome d Terminal Oxidase Complex from Escherichia coli

The cytochrome d terminal oxidase complex is a principal component of the aerobic respiratory chain of Escherichia coli. This purified complex contains two polypeptides as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the reduced minus oxidized spectrum indicates that cytochromes 6-558, a₁, and d are all components of the complex. Changes in the absorption spectrum of the complex between 500 and 700 nm were analyzed as a function of the solution electrochemical potential. A spectral resolution algorithm was used to extract the reduced minus oxidized spectrum of each electrochemically active species from the set of spectra of the complex at different solution potentials. The procedure yielded reduced minus oxidized difference spectra for cytochromes 6-558, a₁ and d, along with the midpoint potentials and Nernst n values for each cytochrome. The midpoint potentials (E_m) and n values for the solubilized complex were as follows: cytochrome b-558, E_m = 61 mV and n = 0.8; cytochrome a₁, E_m= 113 mV and n = 1; cytochrome d, E_m = 232 mV and n = 1. The spectrum of cytochrome 6-558 was typical of 6-type cytochromes, and that of cytochrome d was dominated by a band centered at 628 nm. The difference spectrum of cytochrome a₁, indicated an α band at 594 nm, a strong β band near 560 nm, and a trough near 645 nm. The spectrum is similar to the spectra of high-spin heme a model compounds.