Mutation analysis of the interaction of B-type cytochrome c oxidase with its natural substrate cytochrome c-551(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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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.
著者
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Kabashima Yoshiki
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology
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Sakamoto Junshi
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology
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Ueda Naoaki
Department of Bioscience, and Bioinformatics, Kyushu Institute of Technology
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Sone Nobuhito
Department of Bioscience, and Bioinformatics, Kyushu Institute of Technology
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Ueda Naoaki
Department Of Bioscience And Bioinformatics Kyushu Institute Of Technology
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Sakamoto Junshi
Department Of Bioscience And Bioinformatics Kyushu Institute Of Technology
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Sakamoto Junshi
Department Of Biochemical Engineering And Science Kyushu Institute Of Technology
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Sone Nobuhito
Department Of Bioscience And Bioinformatics Kyushu Institute Of Technology
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Sone Nobuhito
Department Of Biochemical Engineering And Science Faculty Of Computer Science And System Engineering
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Kabashima Yoshiki
Department Of Bioscience And Bioinformatics Kyushu Institute Of Technology
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