Tryptophan 121 of Subunit II Is the Electron Entry Site to Cytochrome-c Oxidase in Paracoccus denitrificans: INVOLVEMENT OF A HYDROPHOBIC PATCH IN THE DOCKING REACTION

Heike Witt,Francesco Malatesta,Flavia Nicoletti,Maurizio Brunori,Bernd Ludwig

doi:10.1074/jbc.273.9.5132

Abstract

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.

Highlights

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase
The involvement of electrostatic interactions in the reaction between cytochrome c and cytochrome-c oxidase mediated by acidic residues predominantly located on subunit II of the P. denitrificans oxidase has already been demonstrated [6]
By applying standard site-directed mutagenesis techniques, we constructed several mutants in subunit II of cytochrome oxidase to investigate the contribution of hydrophobic residues to the interaction with cytochrome c

Summary

Introduction

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. Several studies with cytochrome c and its different redox partners demonstrated the involvement of electrostatic interactions mediated by lysines surrounding the heme edge on cytochrome c and acidic residues on the counterpart [1,2,3,4,5,6]. It became obvious from ionic strength dependence measurements that electrostatic interactions are not the only criterion governing optimal electron transfer [2]. Comparison of the kinetic parameters reveals that the initial binding of cytochrome c, as reflected by KM, is not affected by the mutations but the electron transfer is impaired, suggesting that an optimal configuration cannot be achieved in the mutant enzymes

Methods

Results

Conclusion