Spectroscopic evidence for a heme-heme binuclear center in the cytochrome bd ubiquinol oxidase from Escherichia coli

The cytochrome bd complex is a ubiquinol oxidase, which is part of the aerobic respiratory chain of Escherichia coli. This enzyme is structurally unrelated to the heme-Cu oxidases such as cytochrome c oxidase. While the cytochrome bd complex contains no copper, it does have three heme prosthetic groups: heme b₅₅₈, heme b₅₉₅, and heme d (a chlorin). Heme b₅₅₈ appears to be involved in the oxidation of quinol, and heme d is known to be the site where oxygen binds and is reduced to water. The role of heme b₅₉₅, which is high spin, is not known. In this paper, CO is used to probe the oxygen-binding site by use of Fourier transform infrared spectroscopy to monitor the stretching frequency of CO bound to the enzyme. Photodissociation at low temperature (e.g., 20 K) of the CO-heme d adduct results in CO associated with the protein within the heme binding pocket. This photodissociated CO can subsequently relax to form a kinetically trapped CO-heme b₅₉₅ adduct. The data clearly show that heme d and heme b₅₉₅ must reside within a common binding pocket in the enzyme. The catalytic active site where oxygen is reduced to water is, thus, properly considered to be a heme d-heme b₅₉₅ binuclear center. This is analogous to the heme a₃-Cu_B binuclear center in the heme-Cu oxidases. Heme b₅₉₅ may play roles analogous to those proposed for the Cu_B component of cytochrome c oxidase.