Importance of Hydrophobic Interaction between a SoxB-Type Cytochrome c Oxidase with Its Natural Substrate Cytochrome c-551 and Its Mutants.

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  • Kagekawa Sayaka
    Department of Biochemical Engineering and Science, Kyushu Institute of Technology
  • Mizukami Makoto
    Department of Research and Development, Higeta Shoyu Co.
  • Noguchi Shunsuke
    Department of Biochemical Engineering and Science, Kyushu Institute of Technology
  • Sakamoto Junshi
    Department of Biochemical Engineering and Science, Kyushu Institute of Technology
  • Sone Nobuhito
    Department of Biochemical Engineering and Science, Kyushu Institute of Technology

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説明

Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551. Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their Km values were 0.04-0.12 μM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa3 oxidase has already been established.

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