Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants.

Abstract

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 K(m) values were 0.04-0.12 microM, 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 aa(3) oxidase has already been established.