Inhibition of Complex I by Ca 2+ Reduces Electron Transport Activity and the Rate of Superoxide Anion Production in Cardiac Submitochondrial Particles † Satoshi Matsuzaki and Luke I. Szweda* Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 ReceiVed August 29, 2006; ReVised Manuscript ReceiVed December 5, 2006 ABSTRACT: Declines in the rate of mitochondrial electron transport and subsequent increases in the half- life of reduced components of the electron transport chain can stimulate O 2 •- formation. We have previously shown that, in solubilized cardiac mitochondria, Ca 2+ mediates reversible free radical-induced inhibition of complex I. In the study presented here, submitochondrial particles prepared from rat heart were utilized to determine the effects of Ca 2+ on specific components of the respiratory chain and on the rates of electron transport and O 2 •- production. The results indicate that complex I is inactivated when submitochondrial particles are treated with Ca 2+ . Inactivation was specific to complex I with no alterations in the activities of other electron transport chain complexes. Complex I inactivation by Ca 2+ resulted in the reduction of NADH-supported electron transport activity. In contrast to the majority of electron transport chain inhibitors, Ca 2+ suppressed the rate of O 2 •- production. In addition, while inhibition of complex III stimulated O 2 •- production, Ca 2+ reduced the relative rate of O 2 •- production, consistent with the magnitude of complex I inhibition. Evidence indicates that complex I is the primary source of O 2 •- released from this preparation of submitochondrial particles. Ca 2+ therefore inhibits electron transport upstream of site- (s) of free radical production. This may represent a means of limiting O 2 •- production by a compromised electron transport chain. Mitochondria have long been recognized for their role in the maintenance of cellular energy status and function. Mitochondria are also capable of accumulating Ca 2+ (1-5) and producing oxygen-derived free radicals (6-13). Recent interest in these organelles has been stimulated by their acknowledged role in necrotic and apoptotic cell death. Both forms of cell death are dependent on dramatic changes in mitochondrial function and/or structure and can be initiated by disruption of Ca 2+ and/or redox homeostasis (14-22). Mitochondria are therefore responsive to alterations in Ca 2+ status, produce highly reactive pro-oxidants, and integrate a variety of cellular alterations that ultimately determine the fate of the cell. Important areas of investigation seek to define how these processes are regulated and to identify molecular determinants that shift the balance from normal physiological control to pathophysiological disintegration. An increase in the mitochondrial Ca 2+ concentration can activate the matrix dehydrogenases pyruvate dehydrogenase, R-ketoglutarate dehydrogenase, and NAD + -isocitrate dehy- drogenase (23-31). These stimulatory effects are postulated to couple the energetic demands of muscle contraction and relaxation with ATP synthesis (32). Unabated, mitochondrial Ca 2+ uptake can, however, induce large-amplitude swelling, disruption of mitochondrial integrity and function, cyto- chrome c release, and cellular necrosis and/or apoptosis (4, 15, 17-22). Similarly, free radicals are capable of reversibly altering mitochondrial function. Redox-dependent inhibition of R-ketoglutarate dehydrogenase (33-35) may underlie an antioxidant response limiting reducing equivalents required for the production of superoxide anion and/or protecting key sulfhydryl residues from irreversible oxidative modification (36-38). Oxygen-derived free radical species can, however, induce irreversible inactivation of protein function (39, 40) and irreparable loss of mitochondrial respiratory activity and ATP synthesis (41-44). Evidence indicates that mitochon- drial Ca 2+ uptake and free radical production are interrelated (45). Ca 2+ can induce declines in the rate of electron transport through the disruption of the inner mitochondrial membrane (17-20, 22, 46, 47). Declines in the rate of electron transport and increases in the half-life of reduced components of the electron transport chain can increase the rate of O 2 •- formation (6-13). Conversely, a decrease in the rate of electron transport may diminish the proton gradient and subsequently the rate of uptake of Ca 2+ by the mitochondria (1-5). Reports on the effects of Ca 2+ on mitochondrial pro- oxidant production have varied (2, 20, 48, 49). This is likely due to differences in the experimental model that is utilized, the method of free radical detection, and the manner in which Ca 2+ is administered (20, 45). Our study sought to determine mechanisms by which Ca 2+ alters mitochondrial electron transport and the effects of these alterations on O 2 •- production. Submitochondrial particles isolated from cardiac † This work was supported by grants from the American Heart Association (0625752Z) and the Oklahoma Center for Advancement of Science and Technology (HR05-171S). * To whom correspondence should be addressed: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104. Phone: (405) 271-7582. Fax: (405) 271-1437. E-mail: Luke-Szweda@omrf.ouhsc.edu. 1350 Biochemistry 2007, 46, 1350-1357 10.1021/bi0617916 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/18/2007