Binding of O(2) and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans

Biochemistry. 2000 May 30;39(21):6365-72. doi: 10.1021/bi000123w.


The crystal structure of the heme-copper oxidases suggested a putative channel of oxygen entry into the heme-copper site of O(2) reduction. Changing a conserved valine near this center in cytochrome bo(3) of Escherichia coli to isoleucine caused a significant increase in the apparent K(M) for oxygen with little or no change in V(max), suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., García-Horsman, A., Iwata, S., Michel, H., and Wikström, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa(3) from Paracoccus denitrificans. In this mutant, the apparent K(M) for oxygen is 8 times higher than in the wild-type enzyme, whereas V(max) is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases of the reaction with O(2) are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O(2) entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Conserved Sequence
  • Cytochrome b Group
  • Cytochromes / chemistry
  • Cytochromes / metabolism
  • Electron Transport Complex IV / chemistry*
  • Electron Transport Complex IV / metabolism*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins
  • Heme / analogs & derivatives
  • Heme / chemistry
  • Heme / metabolism
  • Isoleucine*
  • Kinetics
  • Models, Chemical
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Oxygen / metabolism*
  • Paracoccus denitrificans / enzymology*
  • Photolysis
  • Spectrophotometry
  • Spectroscopy, Fourier Transform Infrared
  • Valine*


  • Cytochrome b Group
  • Cytochromes
  • Escherichia coli Proteins
  • cytochrome bo3, E coli
  • Isoleucine
  • heme a
  • Heme
  • Electron Transport Complex IV
  • Valine
  • Oxygen