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1.  Reactive Oxygen Species Production by Forward and Reverse Electron Fluxes in the Mitochondrial Respiratory Chain 
PLoS Computational Biology  2011;7(3):e1001115.
Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.
Author Summary
Respiration at the level of mitochondria is considered as delivery of electrons and protons from NADH or succinate to oxygen through a set of transporters constituting the respiratory chain (RC). Mitochondrial respiration, dealing with transfer of unpaired electrons, may produce reactive oxygen species (ROS) such as O2− and subsequently H2O2 as side products. ROS are chemically very active and can cause oxidative damage to cellular components. The production of ROS, normally low, can increase under stress to the levels incompatible with cell survival; thus, understanding the ways of ROS production in the RC represents a vital task in research. We used mathematical modeling to analyze experiments with isolated brain mitochondria aimed to study relations between electron transport and ROS production. Elsewhere we reported that mitochondrial complex III can operate in two distinct steady states at the same microenvironmental conditions, producing either low or high levels of ROS. Here, this property of bistability was confirmed for the whole RC. The associations between measured ROS production and computed individual free radical levels in complexes I and III were established. The discovered phenomenon of bistability is important as a basis for new strategies in organ transplantation and therapy.
doi:10.1371/journal.pcbi.1001115
PMCID: PMC3068929  PMID: 21483483
2.  Integration of enzyme kinetic models and isotopomer distribution analysis for studies of in situ cell operation 
BMC Neuroscience  2006;7(Suppl 1):S7.
A current trend in neuroscience research is the use of stable isotope tracers in order to address metabolic processes in vivo. The tracers produce a huge number of metabolite forms that differ according to the number and position of labeled isotopes in the carbon skeleton (isotopomers) and such a large variety makes the analysis of isotopomer data highly complex. On the other hand, this multiplicity of forms does provide sufficient information to address cell operation in vivo. By the end of last millennium, a number of tools have been developed for estimation of metabolic flux profile from any possible isotopomer distribution data. However, although well elaborated, these tools were limited to steady state analysis, and the obtained set of fluxes remained disconnected from their biochemical context. In this review we focus on a new numerical analytical approach that integrates kinetic and metabolic flux analysis. The related computational algorithm estimates the dynamic flux based on the time-dependent distribution of all possible isotopomers of metabolic pathway intermediates that are generated from a labeled substrate. The new algorithm connects specific tracer data with enzyme kinetic characteristics, thereby extending the amount of data available for analysis: it uses enzyme kinetic data to estimate the flux profile, and vice versa, for the kinetic analysis it uses in vivo tracer data to reveal the biochemical basis of the estimated metabolic fluxes.
doi:10.1186/1471-2202-7-S1-S7
PMCID: PMC1775047  PMID: 17118161

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