Control of mitochondrial superoxide production by reverse electron transport at complex I
The generation of mitochondrial superoxide (O 2 . ) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H 2 O 2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O 2 . production by RET in isolated heart mitochondria. Measuring mitochondrial H 2 O 2 production at a range of proton-motive force (p) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O 2 . production by RET responds to changes in O 2 concentration, the magnitude of p, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O 2 . production at complex I, underscoring the importance of the CoQ pool for mitochondrial O 2 . production by RET. An analysis of O 2 . production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O 2 . production in both pathological and physiological conditions. We conclude that O 2 . production by RET is highly responsive to small changes in p and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling. Superoxide (O 2 . ) 3 is the proximal reactive oxygen species (ROS) formed within mitochondria, with most O 2 . being very rapidly converted to H 2 O 2 by manganese superoxide dismutase (MnSOD) within the matrix (1, 2). As well as contributing to oxidative damage, H 2 O 2 acts as a redox signal, both within the mitochondria and in the cytosol (3-6). This mode of signal transduction arises via the reversible oxidation of protein thiols that pass on the modification to effector proteins as a redox relay (3-6). Whereas there are a number of potential mitochondrial sources of O 2 .