Aguirre 2010 Biochim Biophys Acta
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Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim Biophys Acta 1797:557-65. |
Aguirre E, Rodriguez-Juarez F, Bellelli A, Gnaiger E, Cadenas S (2010) Biochim Biophys Acta
Abstract: Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O_{2} concentrations and mitochondrial O_{2} consumption by high-resolution respirometry over a range of O_{2} concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC_{50} on [O_{2}] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O_{2} and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O_{2} dependent oxidation. It also explains the non-linear dependence of IC_{50} on O_{2} concentration, and the hyperbolic increase of c_{50} as a function of NO concentration.
• Keywords: Nitric oxide, Mitochondrial respiration, Cytochrome c oxidase, Oxygen consumption, Mitochondria, Kinetic model
• O2k-Network Lab: ES Madrid Cadenas S, AT Innsbruck Gnaiger E
Labels: MiParea: Respiration, Genetic knockout;overexpression
Stress:Oxidative stress;RONS
Tissue;cell: HEK Preparation: Intact cells, Enzyme, Oxidase;biochemical oxidation Enzyme: Complex IV;cytochrome c oxidase Regulation: Inhibitor, Oxygen kinetics Coupling state: LEAK, ROUTINE, ETS Pathway: ROX HRR: Oxygraph-2k, NO
Contents |
Supporting Information
The hyperbolic approximation
The kinetic model described in this manuscript can be simplified, under selected sets of experimental conditions, in order to provide hyperbolic approximations valid within certain ranges of O_{2} and NO concentration.
We first need to consider the general equation for the velocity of the O_{2} consumption catalyzed by CcO:
v = [CcO_{tot}] V_{max1} ([O_{2}] K_{m2} K_{icNO} K_{uNO} + r [O_{2}] [NO] K_{m1} K_{uNO}) / [K_{m2} K_{icNO} K_{uNO} (K_{m1} + [O_{2}]) + [NO] K_{m1} K_{uNO} (K_{m2} + [O_{2}]) + [NO] [O_{2}] K_{m2} K_{icNO}] Eq. (S1)
Note that this equation is, as expected, the weighted sum of two Michaelis cycles plus the term [NO] [O_{2}] K_{m2} K_{icNO} which represents the fully inhibited species NO CcO_{o}.
Under our experimental conditions K_{uNO} is larger than the concentrations of NO, and is
thus dropped by the minimization routine; this implies that the pertinent enzyme derivative, NO CcO_{o}, is not populated and that we can omit the term K_{uNO}. Thus the velocity of O_{2} consumption reduces to
v = [CcO_{tot}] V_{max1} ([O_{2}] K_{m2} K_{icNO} + r [O_{2}] [NO] K_{m1}) / [K_{m2} K_{icNO} (K_{m1} + [O_{2}]) + [NO] K_{m1} (K_{m2} + [O_{2}])] Eq. (S2)
Eq. S2 reduces to two simple hyperbola in the absence of NO or in the presence of excess NO (i.e. when [NO] K_{m1} >> K_{m2} K_{icNO}).
Rearranging from Eq. 2 in the main text, the c_{50} derived from Eq. S2 is as follows:
c_{50} = K_{m1} K_{m2} [K_{icNO} / (K_{icNO} K_{m2} + [NO] K_{m1}) + [NO] / (K_{icNO} K_{m2} + [NO] K_{m1})] Eq. (S3)
This equation shows that the c_{50} is limited between K_{m1} (in the absence of NO) and K_{m2} (at very high NO), and approximates as follows:
c_{50} = K_{m1} K_{m2} K_{icNO} / (K_{icNO} K_{m2} + [NO] K_{m1}) at low NO; and
c_{50} = K_{m1} K_{m2} [NO] / (K_{icNO} K_{m2} + [NO] K_{m1}) at high NO.
If we take K_{m1} as granted from the experiments in the absence of NO, these approximations allow us to determine empirically the two products K_{m1} K_{m2} and K_{icNO} K_{m2}.
With K_{m1} = 0.81 μM, V_{max1} =16.5 pmol·s^{-1}·10^{-6} cells, K_{icNO} = 3.63 nM and K_{m2} = 520 μM, the hyperbolic approximation would suggest V_{max2} = 20.4 pmol·s^{-1}·10^{-6} cells (kinetic fit = 22) and K_{uNO} = 9.94 μM (compatible with the kinetic fit since K_{uNO}>[NO]); K_{m1} K_{m2} = 421 μM and K_{icNO} K_{m2} = 1.89 μM.
For K_{m1} = 0.81 μM, the hyperbolic approximation yields the best global fit to the low-O2 experimental series when K_{icNO} = 3.37 nM, K_{m2} = 407 μM and K_{uNO} = 7.23 μM; K_{m1} K_{m2} = 330 μM and K_{icNO} K_{m2} = 1.37 μM.
For K_{m1} = 0.65 μM, which corresponds to the average c_{50} measured in the absence of NO (Table 1), the hyperbolic approximation yields the best global fit to the low-O2 experimental series when K_{icNO} = 2.6 nM, K_{m2} = 476 μM and K_{uNO} = 3.54 μM; K_{m1} K_{m2} = 309 μM and K_{icNO} K_{m2} = 1.24 μM.
The parameters for the hyperbolic approximation were calculated with variable J_{S} for each experimental run. The stimulation factor, F, was calculated from the J_{S}/J_{ref} ratio for each experiment (Fig. 7C and D).
Parameters in the kinetic and hyperbolic models
Kinetic Parameter
K_{m1}=V_{max1}/V_{max2} = 0.810
V_{max1} = 16.500
V_{max2}=V_{max1}/K_{m1} = 20.370
r=1/K_{m1} = 1.235
K_{m*}=K_{m1*} K_{m2} = 421.200
K_{m2}=K_{m*}/K_{m1} = 520.000
K_{icNO} = 0.00363
K_{i*}=K_{icNO*} K_{m2} = 1.888
K_{uNO}=K_{i*}/(1-K_{m1}) = 9.935
Hyperbolic Parameter 1 or (2)
K_{m1}=V_{max1}/V_{max2} = 0.650 (0.810)
V_{max1} --
V_{max2}=V_{max1}/K_{m1} --
r=1/K_{m1} = 1.538 (1.235)
K_{m*}=K_{m1*} K_{m2} = 309.350 (330.000)
K_{m2}=K_{m*}/K_{m1} = 475.923 (407.407)
K_{icNO} = 0.00260 (0.00337)
K_{i*}=K_{icNO*} K_{m2} = 1.237 (1.373)
K_{uNO}=K_{i*}/(1-K_{m1}) = 3.535 (7.226)
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