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 structu rally 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 pape r, CO is used to probe the oxygen-binding site by use of Fourier trans form infrared spectroscopy to monitor the stretching frequency of CO b ound to the enzyme. Photodissociation at low temperature (e.g., 20 K) of the CO-heme d adduct results in CO associated with the protein with in the heme binding pocket. This photodissociated CO can subsequently relax to form a kinetically trapped CO-heme b595 adduct. The data clea rly show that heme d and heme b595 must reside within a common binding pocket in the enzyme. The catalytic active site where oxygen is reduc ed to water is, thus, properly considered to be a heme d-heme b595 bin uclear 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.