TRYPTOPHAN-136 IN SUBUNIT-II OF CYTOCHROME-BO(3) FROM ESCHERICHIA-COLI MAY PARTICIPATE IN THE BINDING OF UBIQUINOL

In the cytochrome c oxidases, the role of subunit II is to provide the electron entry site into the enzyme. This subunit contains both the b inding site for the substrate, cytochrome c, and the CUA redox center, which is initially reduced by cytochrome c, Cytochrome bo(3) and othe r quinol oxidases that are members of the heme-copper oxidase superfam ily have a homologous subunit II, but the Cu-A Site is absent, as is t he docking site for cytochrome c. Speculation that subunit II in the q uinol oxidases may also be important as an electron entry site is supp orted by the demonstration several years ago that a photoreactive subs trate analogue, azido-Q, covalently labeled subunit II in cytochrome b o(3). In the current work, a sequence alignment of subunit II of heme- copper quinol oxidases is used as a guide to select conserved residues that might be important for the binding of ubiquinol to cytochrome bo (3). Results are presented for point mutants in 24 different residue p ositions in subunit II. The membrane-bound enzymes were examined by op tical spectroscopy and by determining the activity of ubiquinol-l oxid ase. In each case, the K-m for ubiquinol-l was determined as a measure of possible perturbation to a quinol binding site. The only mutant th at had a noticeably altered K-m for ubiquinol-l was W136A, in which th e K-m was about sixfold increased. Thus, W136 may be at or close to a substrate (ubiquinol)-binding site in cytochrome bo(3). In the cytochr ome c oxidases, the equivalent tryptophan (W121 in Paracoccus denitrif icans) has been identified as the ''electron entry site''.