Aguirre 2010 Biochim Biophys Acta
|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.|
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 O2 concentrations and mitochondrial O2 consumption by high-resolution respirometry over a range of O2 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 IC50 on [O2] 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 O2 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 O2 dependent oxidation. It also explains the non-linear dependence of IC50 on O2 concentration, and the hyperbolic increase of c50 as a function of NO concentration.
Labels: MiParea: Respiration, Genetic knockout;overexpression Cell line: HEK Preparation: Intact cells, Enzyme, Oxidase;biochemical oxidation Enzyme: Complex IV;cytochrome c oxidase Stress: Oxidative stress;RONS Regulation: Inhibitor, Oxygen kinetics Coupling state: LEAK, ROUTINE, ETS Substrate state: ROX HRR: Oxygraph-2k, NO
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 O2 and NO concentration.
We first need to consider the general equation for the velocity of the O2 consumption catalyzed by CcO:
v = [CcOtot] Vmax1 ([O2] Km2 KicNO KuNO + r [O2] [NO] Km1 KuNO) / [Km2 KicNO KuNO (Km1 + [O2]) + [NO] Km1 KuNO (Km2 + [O2]) + [NO] [O2] Km2 KicNO] Eq. (S1)
Note that this equation is, as expected, the weighted sum of two Michaelis cycles plus the term [NO] [O2] Km2 KicNO which represents the fully inhibited species NO CcOo.
Under our experimental conditions KuNO is larger than the concentrations of NO, and is thus dropped by the minimization routine; this implies that the pertinent enzyme derivative, NO CcOo, is not populated and that we can omit the term KuNO. Thus the velocity of O2 consumption reduces to
v = [CcOtot] Vmax1 ([O2] Km2 KicNO + r [O2] [NO] Km1) / [Km2 KicNO (Km1 + [O2]) + [NO] Km1 (Km2 + [O2])] 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] Km1 >> Km2 KicNO).
Rearranging from Eq. 2 in the main text, the c50 derived from Eq. S2 is as follows:
c50 = Km1 Km2 [KicNO / (KicNO Km2 + [NO] Km1) + [NO] / (KicNO Km2 + [NO] Km1)] Eq. (S3)
This equation shows that the c50 is limited between Km1 (in the absence of NO) and Km2 (at very high NO), and approximates as follows:
c50 = Km1 Km2 KicNO / (KicNO Km2 + [NO] Km1) at low NO; and
c50 = Km1 Km2 [NO] / (KicNO Km2 + [NO] Km1) at high NO.
If we take Km1 as granted from the experiments in the absence of NO, these approximations allow us to determine empirically the two products Km1 Km2 and KicNO Km2.
With Km1 = 0.81 μM, Vmax1 =16.5 pmol·s-1·10-6 cells, KicNO = 3.63 nM and Km2 = 520 μM, the hyperbolic approximation would suggest Vmax2 = 20.4 pmol·s-1·10-6 cells (kinetic fit = 22) and KuNO = 9.94 μM (compatible with the kinetic fit since KuNO>[NO]); Km1 Km2 = 421 μM and KicNO Km2 = 1.89 μM.
For Km1 = 0.81 μM, the hyperbolic approximation yields the best global fit to the low-O2 experimental series when KicNO = 3.37 nM, Km2 = 407 μM and KuNO = 7.23 μM; Km1 Km2 = 330 μM and KicNO Km2 = 1.37 μM.
For Km1 = 0.65 μM, which corresponds to the average c50 measured in the absence of NO (Table 1), the hyperbolic approximation yields the best global fit to the low-O2 experimental series when KicNO = 2.6 nM, Km2 = 476 μM and KuNO = 3.54 μM; Km1 Km2 = 309 μM and KicNO Km2 = 1.24 μM.
The parameters for the hyperbolic approximation were calculated with variable JS for each experimental run. The stimulation factor, F, was calculated from the JS/Jref ratio for each experiment (Fig. 7C and D).
Parameters in the kinetic and hyperbolic models
Km1=Vmax1/Vmax2 = 0.810
Vmax1 = 16.500
Vmax2=Vmax1/Km1 = 20.370
r=1/Km1 = 1.235
Km*=Km1* Km2 = 421.200
Km2=Km*/Km1 = 520.000
KicNO = 0.00363
Ki*=KicNO* Km2 = 1.888
KuNO=Ki*/(1-Km1) = 9.935
Hyperbolic Parameter 1 or (2)
Km1=Vmax1/Vmax2 = 0.650 (0.810)
r=1/Km1 = 1.538 (1.235)
Km*=Km1* Km2 = 309.350 (330.000)
Km2=Km*/Km1 = 475.923 (407.407)
KicNO = 0.00260 (0.00337)
Ki*=KicNO* Km2 = 1.237 (1.373)
KuNO=Ki*/(1-Km1) = 3.535 (7.226)