# 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. |

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, ET
**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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