Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells.
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.
Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fenández Almagro 3, Madrid, Spain.
Enara
E
Aguirre
Félix
F
Rodríguez-Juárez
Andrea
A
Bellelli
Erich
E
Gnaiger
Susana
S
Cadenas
D019069:Cell Respiration; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000201:enzymology; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption
D016166:Free Radical Scavengers; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D003576:Electron Transport Complex IV; D010100:Oxygen