Mutation analysis of the interaction of B-type cytochrome c oxidase with its natural substrate cytochrome c-551.
Heme-copper oxidases in the respiratory chain are classified into three subfamilies: A-, B- and C-types. Cytochrome bo(3)-type cytochrome c oxidase from thermophilic Bacillus is a B-type oxidase that is thought to interact with cytochrome c through hydrophobic interactions. This is in contrast to A-type oxidases, which bind cytochrome c molecules primarily through electrostatic forces between acidic residues in the oxidase subunit II and basic residues within cytochromes. In order to investigate the substrate-binding site in cytochrome bo(3), eight acidic residues in subunit II were mutated to corresponding neutral residues and enzymatic activity was measured using cytochrome c-551 from closely related Bacillus PS3. The mutation of E116, located at the interface to subunit I, decreased the k(cat) value most prominently without affecting the K(m) value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the Cu(A) site, largely affected both values, suggesting that it is important for both electron transfer and substrate binding. The mutation of D49 and E84 did not affect enzyme kinetic parameters, but the mutation of E64, E66 and E68 lowered the affinity of cytochrome bo(3) for cytochrome c-551 without affecting the k(cat) value. These three residues are located at the front of the hydrophilic globular domain and distant from the Cu(A) site, suggesting that these amino acids compose an acidic patch for a second substrate-binding site. This is the first report on site-directed mutagenesis experiments of a B-type heme-copper oxidase.
Kabashima
Yoshiki
Y
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
Ueda
Naoaki
N
Sone
Nobuhito
N
Sakamoto
Junshi
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D056506:Geobacillus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C017167:cytochrome C(551); D003576:Electron Transport Complex IV