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Kinetics of Ubiquinol Cytochrome c Reductase: Lack of a Diffusion-Limited Step with Short Chain Ubiquinols as Substrates

  • R. Fato
  • C. Castelluccio
  • G. Lenaz
  • M. Battino
  • G. Parenti Castelli

Abstract

The steady-state kinetics of mitochondrial ubiquinol-cyt.c reductase has been investigated both in submitochondrial particles (SMP) and in the isolated bc1 complex using a new method to determine the concentrations of ubiquinol in the lipid bilayer. The method, involving saturation kinetics determinations at varying membrane phospholipid fractional volumes α at saturating cyt.c, allows to simultaneously evaluate the real Km of the enzyme for ubiquinol-1 and -2 dissolved in the lipid and their partition coefficients P, according to:
$${K_m}(app)\, = \alpha .\,\left( {{K_m}\, - \,{K_m}/P} \right)\, + \,{K_m}/P$$
where Km(app) is the apparent Km in the total assay mixture and Km is expressed in mol/liter of lipid phase. Increases of phospholipid in the medium decrease the actual QH2 concentration in the lipids and therefore increase Km(app). A plot of Km(app) vs. α allows determination of both P and Km. Table I shows that different Km(app) correspond to almost identical Km, a consequence of the different P. The Km of duroquinol is independent of lipid concentration, indicating that it may interact with a different mechanism. The kcat are independent of α, and much higher with ubiquinol-2 than -1. It is also clear that the minimal second-order rate constant for ubiquinol oxidation, kcat/Km, increases from ubiquinol-1 to -2.

Keywords

Partition Coefficient Lipid Bilayer Lipid Concentration Lipid Phase Order Rate Constant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    A.R. Crofts (1986) J. Bioenerg. Biomembr. JJS, 437–451.Google Scholar
  2. 2.
    C.R. Hackenbrock, B. Chazotte, and S.S. Gupte (1986) J. Bioenerg. Biomembr. 18, 331–368.CrossRefGoogle Scholar
  3. 3.
    R. Fato, M. Battino, M. Degli Esposti, G. Parenti Castelli, and G. Lenaz (1986) Biochemistry 25, 3378–3390.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • R. Fato
    • 1
  • C. Castelluccio
    • 1
  • G. Lenaz
    • 1
  • M. Battino
    • 2
  • G. Parenti Castelli
    • 2
  1. 1.Department of BiologyUniversity of BolognaBolognaItaly
  2. 2.Department of BiochemistryUniversity of BolognaBolognaItaly

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