IDENTIFICATION OF A NOVEL QUINONE-BINDING SITE IN THE CYTOCHROME BO COMPLEX FROM ESCHERICHIA-COLI

The cytochrome bo complex is a heme BO-type heme-copper quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as an electron transfer-linked proton pump. To study the protein-mediate d electron transfer from substrates to metal centers, we carried out q uantitative and qualitative analyses of a bound quinone in the purifie d oxidase and found that it has a novel high affinity ubiquinone-bindi ng site distinct from the quinol oxidation site. Enzymatic and spectro scopic studies suggest that the quinone-binding site is located close to both the quinol oxidation site in subunit II and low-spin heme B in subunit I. The quinone-binding site of a bound ubiquinone-free oxidas e was reconstituted with the potent quinol oxidation site inhibitor 2, 6-dichloro-4-nitrophenol, which decreased the V-max value of the ubiqu inol-1 oxidase activity to one-fourth of the control activity. These r esults indicate that the quinone-binding site is essential for the cat alytic functions of the cytochrome bo complex and mediates electron tr ansfer from the quinol oxidation site to the low-spin heme.