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 bo3-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 bo3, 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 kcat value most prominently without affecting the Km value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the CuA 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 bo3 for cytochrome c-551 without affecting the kcat value. These three residues are located at the front of the hydrophilic globular domain and distant from the CuA 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.