Contents

๐Ÿ“’ Krab 2011

Explaining the enigmatic KMfor oxygen in cytochrome c oxidase: A kinetic model1

Sciwheel

Introduction

  • Complex IV (cytochrome c oxidase) reaction: 4cytc2+ + 8H_M+ + O2 โ†’ 4cytc3+ + 4H_C+ + 2H2O

  • Apparent high affinity (low Km,app) for oxygen (<1ฮผM). But the dissociation constant is 0.28mM?! => fast trapping of the already bound O2

  • High pmf or low c2+/c3+: higher Km,app = 1~10 ฮผM

  • Kinetic models for this enzyme that incorporate all features of Gibbs energy transduction are scarce

The model

The reaction cycle

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr1_lrg.jpg https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-fx1.jpg

https://user-images.githubusercontent.com/40054455/86699812-2091f680-c043-11ea-9495-daeb03faa30d.png https://user-images.githubusercontent.com/40054455/86699816-212a8d00-c043-11ea-98a4-2c45c20509dc.png

  • The number of charges transported across the inner mitochondrial membrane is given in parentheses.
  • Division of reaction 5, the O to E transition, into 6 steps to describe the proton pumping mechanism https://user-images.githubusercontent.com/40054455/86699819-21c32380-c043-11ea-95df-7d524eb59ab1.png https://user-images.githubusercontent.com/40054455/86699820-225bba00-c043-11ea-8323-b73e0388c648.png https://user-images.githubusercontent.com/40054455/86699823-225bba00-c043-11ea-8602-1a3842dc463b.png https://user-images.githubusercontent.com/40054455/86699825-22f45080-c043-11ea-9a9c-29c44be77c6e.png https://user-images.githubusercontent.com/40054455/86699827-238ce700-c043-11ea-9387-0d5f0355f0ba.png https://user-images.githubusercontent.com/40054455/86699829-238ce700-c043-11ea-97dc-2c49a4507b27.png
  • 6-fold divisions of reactions 3, 4 and 6 are also modelled on this

Values of model parameters

https://user-images.githubusercontent.com/40054455/86699844-2687d780-c043-11ea-854e-72351e43e2c9.png

Variables in the model

  • fractions for cytc2+ vs cytc3+
  • free proton concentrations at the C-side and M-side
  • transmembrane electric potential difference (C-side minus M-side): membrane potential dependence of both kinetic constants is equal but opposite $$ k_{\text { forward }}=k_{\text { forward }}^{\circ} \cdot e^{\frac{-q \cdot F \cdot \Delta \psi}{2 R \cdot T}} $$ $$ k_{\mathrm{backward}}=k_{\mathrm{backward}}^{\circ} \cdot e^{\frac{q F \Delta \psi}{2 R T}} $$
    • q2 = 0.33, q3 = 0.67

Calculation of steady state

https://user-images.githubusercontent.com/40054455/86699830-24257d80-c043-11ea-8bc6-b88262bb5f62.png

  • King and Altman method
  • 26 * 26 matrix for transitions rates

Calculation of the KM for O2

  • R00 -> A00 is proportional to [O2]

  • cycle flux to [O2] $$ v=\frac{\alpha \cdot\left[O_{2}\right]-\beta}{\gamma \cdot\left[O_{2}\right]+\delta} $$

  • ฮฒ « ฮฑ โ‹… [O2] $$ v \approx \frac{V_{\max } \cdot\left[O_{2}\right]}{\left[O_{2}\right]+K_{\mathrm{M}}} $$

$$ V_{\max }=\frac{\alpha}{\gamma} \quad K_{\mathrm{M}}=\frac{\delta}{\gamma} $$

Control coef fi cients

$$ C_{i}^{Z}=\frac{\partial \ln Z}{\partial \ln \lambda_{i}} $$

  • ฮป_i is the factor by which both forward and backward rate constant of step i are changed simultaneously
  • this modulation does not alter the ฮ” G0

Results and discussion

Rate and enzyme states as a function of the energy state of the membrane

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr2_lrg.jpg

  • state E04 accumulates (especially under low energy conditions)

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr3_lrg.jpg

  • depends on pmf, absolute H+, ฮ”pH, cytochrome c redox state

Energy state and KM for O2

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr4_lrg.jpg

  • KM values are less than 1 ฮผM at lower values of ฮ”ฮผH+, increase into the micromolar range when ฮ”ฮผH+ is increased
  • a lower reduction level of cytochrome c brings about an increase in KM
  • as in experiments

Control of cycle flux and kinetic properties by individual steps in the mechanism

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr5_lrg.jpg

  • there is no individual step limiting turnover
  • the same steps (13, 14, 17, 19, 25 and 26) contribute signi fi cantly to control of all three of these entities,

https://ars.els-cdn.com/content/image/1-s2.0-S0005272810008029-gr6_lrg.jpg

  • 8 main contributions (control values > 0.05) to control of KMas a function of energy state ( ฮ”ฮผH+)

https://user-images.githubusercontent.com/40054455/86699842-25ef4100-c043-11ea-9819-354bb9e48e2f.png

  • Apart from the R00โ†’ A00and A00โ†’ P00transitions, all these steps are electrogenic proton movements

Sensitivity of KMincrease to arbitrarily chosen parameters

  • The main prediction of our model is the increase of KMfor oxygen upon energisation
  • A number of parameters in the model (midpoint potentials of heme a3 in the P, F, E states, pKaof protonation of the Fe-ligand in the F, E states and 6 bias factors) have arbitrarily assigned values.
  • explored how dependent the KMchange predicted by the model is on the values of these parameters, and on the charge separation parameters q2and q3
  • these small variations of the parameter values did not abolish the KMdifference

Utility of the model

  • dependence of the electron transport rate through cytochrome oxidase on membrane energisation is modelled realistically and explicitly.
  • analysis and prediction of the separate effects of membrane potential, matrix pH and intermembrane space pH on the rate
  • Not included: branches of catalytic function (slips), dimerization, regulations
  • It is possible to use this model to calculate transient kinetics of the enzyme

Reference


  1. Krab K, Kempe H, Wikstrรถm M. Explaining the enigmatic K(M) for oxygen in cytochrome c oxidase: a kinetic model. Biochim. Biophys. Acta 2011;1807(3):348-358. doi:10.1016/j.bbabio.2010.12.015. ↩︎