0026-8933/01/3506- $25.00 © 2001 MAIK “Nauka / Interperiodica” 0940 Molecular Biology, Vol. 35, No. 6, 2001, pp. 940–949. Translated from Molekulyarnaya Biologiya, Vol. 35, No. 6, 2001, pp. 1095–1104. Original Russian Text Copyright © 2001 by Demin, Goryanin, Kholodenko, Westerhoff. INTRODUCTION The respiratory chain and O 2 as a final acceptor of electrons increase the number of ATP molecules pro- duced per one and the same amount of substrate [1]. However, such a mechanism sharply enhancing the efficiency is a cause of increased danger. The aggre- gate of redox reactions called respiratory chain includes intermediates capable of easily passing one electron to molecular oxygen, generating superoxide [2]. It is thought that at least two enzymes of the respiratory chain in mitochondria (NADH– ubiquinone reductase, complex I, and ubiquinol–cyto- chrome c reductase, the bc 1 complex) are able to gen- erate superoxide [3–6]. Among the intermediates of reactions catalyzed by complexes I and bc 1 , semi- quinone possesses such an ability. Its concentration and, therefore, the rate of production depends on the membrane potential ∆Ψ , i.e., on the state of the O 2 . – O 2 . – mitochondrion. In the present work, we consider only the superoxide generation by the bc 1 complex. Earlier [3], we observed the correlation between the rate of superoxide generation and ∆Ψ . However, this has been done within a model for one enzyme (the bc 1 complex) and was a result of varying the potential as a parameter. Indeed, the potential is established as a result of mutual work of both the producers (the bc 1 complex) and the consumers (e.g., ade- nine nucleotide translocator and ATP syntase). Taking into account the fact that the membrane permeability for ions also depends (and nonlinearly) on ∆Ψ , one can conclude that to clarify the dependence of the rate of superoxide production on ∆Ψ and to determine the mechanism of its regulation, a more complete descrip- tion of energy transformation in mitochondria is required. ∆μ H + ∆μ H + Kinetic Modeling of Energy Metabolism and Superoxide Generation in Hepatocyte Mitochondria O. V. Demin 1 , I. I. Goryanin 2 , B. N. Kholodenko 3 , and H. V. Westerhoff 4, 5 1 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899 Russia; E-mail: demin@genebee.msu.su 2 Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, SG1 2NY, UK 3 Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA 4 Department of Microbial Physiology, Vrije University, 1081 HV Amsterdam, The Netherlands 5 E.C. Slater Institute, BioCentrum Amsterdam, University of Amsterdam, The Netherlands Received April 3, 2001 Abstract—Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals ( ) in mitochondria. Using all the known data on hepatocyte mitochondria, we have revealed the cor- relation between the rate of superoxide generation by the bc 1 complex and the transmembrane potential (∆Ψ). Assuming that the main electrogenic stage of the Q cycle is the electron transfer between the cytochrome b hemes, then the rate of superoxide generation sharply increases when ∆Ψ grows from 150 to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K + ), the rate of production remains con- stant, although the comparison of the rates at the same ∆Ψ reveals the effect of phosphate or potassium. It turned out that the rate of generation is a function of rather than any of its components. Phosphate and K + have practically no influence on , since the change in ∆Ψ is compensated by ∆pH. The rate of superoxide generation by the bc 1 complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and the oxygen concentration. The kinetic model proposed in this work may serve to understand how the superoxide production is regulated. Key words: superoxide radical, kinetic model, regulation O 2 . – O 2 . – O 2 . – ∆μ H ∆μ H UDC 577.2