Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Myat T. Lin, Robert B. Gennis

Research output: Contribution to journalArticle


Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.

Original languageEnglish (US)
Pages (from-to)1894-1900
Number of pages7
JournalBiochimica et Biophysica Acta - Bioenergetics
Issue number10
StatePublished - Oct 1 2012



  • Cytochrome c
  • Cytochrome c oxidase
  • Kinetics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this