Tryptophan-136 in subunit II of cytochrome bo3 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 binding site for the substrate, cytochrome c, and the CuA redox center, which is initially reduced by cytochrome c. Cytochrome bo3 and other quinol oxidases that are members of the heme-copper oxidase superfamily have a homologous subunit II, but the CuA site is absent, as is the docking site for cytochrome c. Speculation that subunit II in the quinol oxidases may also be important as an electron entry site is supported by the demonstration several years ago that a photoreactive substrate analogue, azido-Q, covalently labeled subunit II in cytochrome bo3. 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 bo3. Results are presented for point mutants in 24 different residue positions in subunit II. The membrane-bound enzymes were examined by optical spectroscopy and by determining the activity of ubiquinol-1 oxidase. In each case, the Km for ubiquinol-1 was determined as a measure of possible perturbation to a quinol binding site. The only mutant that had a noticeably altered Km for ubiquinol-1 was W136A, in which the Km was about sixfold increased. Thus, W136 may be at or close to a substrate (ubiquinol)-binding site in cytochrome bo3. In the cytochrome c oxidases, the equivalent tryptophan (W121 in Paracoccus denitrificans) has been identified as the "electron entry site".
Ma
J
J
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Puustinen
A
A
Wikström
M
M
Gennis
R B
RB
D000595:Amino Acid Sequence; D001386:Azides; D001665:Binding Sites / Q000235:genetics; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels; D017494:Proton Pumps / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D001386:Azides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D019920:Photoaffinity Labels; D017494:Proton Pumps; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; D014364:Tryptophan; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; D010088:Oxidoreductases; C020094:duroquinol oxidase; C003741:ubiquinol