NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated bypass of mitochondrial electron transport chain with artificial and endogenous substrates

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Abstract A wide range of rare and common diseases are linked to mitochondrial dysfunction and associated redox imbalance. Restoration of the underlying redox imbalance by decreasing the cellular NADH/NAD+ ratio could be seen as an extremely useful generalizable strategy in the context of multiple disease states. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a soluble cytoplasmic enzyme that has been mostly viewed as a xenobiotic- metabolizing enzyme, or a bioactivator of cancer drugs at the expense of reducing equivalents of NAD(P)H. Interestingly, some of NQO1 artificial substrates, mostly naphthoquinones, when reduced are capable of subsequently donating their electrons to the mitochondrial electron transport chain downstream of Complex I. This NQO1-mediated alternative electron transfer is therefore an attractive strategy to alleviate reductive stress and support ATP homeostasis as it depends on an endogenous enzyme and only requires addition of respective naphthoquinones. However, naphthoquinones capable of being reduced by NQO1 are either natural products or synthetic redox scaffolds (e.g. idebenone), and we currently lack information on endogenous substrates of NQO1 and its place in cellular redox metabolism. To close this knowledge gap, we will use activity-based metabolomic profiling with recombinant NQO1 to identify cellular endogenous metabolites that are interconverted by this enzyme. Next, we will reconstitute the NQO1-mediated electron transfer with various naphthoquinones in isolated mitochondria and will study the bioenergetics of this non-canonical point of entry of reducing equivalents. This will allow us to rigorously characterize naphthoquinones and related redox-active molecules for their ability to safely bypass a corrupted mitochondrial electron transport chain without inducing oxidative stress. Our current approach will, for the first time, allow us to identify physiological NQO1 substrates and help us better reconstruct the NQO1-mediated electron transfer. This work will ultimately pave the way for developing therapeutic modalities that are based on redox-active small molecules that can alleviate reductive stress.