Superoxide formation at the Q‐binding site of mitochondrial complex I does not arise from a semiquinone that has direct access to the bulk membrane QH 2 /Q pool

Complex I (NADH:Q oxidoreductase) can form superoxide during either forward (NADH oxidizing) electron flux or, under conditions of sufficiently high proton motive force (PMF), reverse electron transport (NAD + reducing). In the isolated enzyme, the fully reduced flavin produces superoxide (site I F ). With mitochondria, maximal superoxide production from site I F (with rotenone) is far less than during reverse electron transport driven by succinate. The marked discrepancy between these two ‘maximal’ rates is consistent with a 2‐site model of superoxide generation. The reduction state of this second site (site I Q ) of superoxide production is unknown, but is often considered a semiquinone. In this study, we determine if superoxide production during complex I reverse electron transport arises from a partially or fully reduced site. We designed the following system to titrate the reduction state of the superoxide generating site: a constant PMF generated by ATP, succinate to reduce the Q pool and inhibitors of complex III isolate the QH 2 /Q from oxidation via the Q‐cycle. Under these conditions, a stepwise oxidation of the QH 2 /Q by malonate titration showed no evidence for the formation a semiquinone, or other partially reduced species, that directly interacts with the bulk Q pool. These titrations suggest, unexpectedly, that superoxide production by site I Q is also by a fully reduced species, although we cannot distinguish if this is a fully reduced single or double electron carrier. Funded by NIH (ARRA) grant number: NIH R01 AG033542