Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition

Abstract

Provision of NAD for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1 and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.

Idebenone Has Distinct Effects on Mitochondrial Respiration in Cortical Astrocytes Compared to Cortical Neurons Due to Differential NQO1 Activity

“…Unfortunately, we saw the Complex I bypass activity of Me-MNQ in cortical astrocytes but not neurons, as was the case for idebenone. This finding suggests that the bypass activity of naphthoquinone derivatives may also require NQO1-reducing activity, as MNQ is a preferred NQO1 substrate (Ravasz et al, 2018). Unlike idebenone, we found that the naphthoquinones MNQ and Me-MNQ increase antimycin A-insensitive oxygen consumption, likely due to superoxide generation, which may limit their therapeutic potential.…”

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

“…To our knowledge, this is the first time that endogenous CoQ has been shown to regulate NOX4 activity, which is in agreement with previous findings for synthetic quinone compounds and NQO1 overexpression [ 18 ]. Importantly, although NQO1 is known to regulate mitochondrial function, its localization on the mitochondrial membrane remains controversial [ [49] , [50] , [51] , [52] , [53] ]. While NQO1 can be detected in mitochondria isolated from multiple mouse tissues, it was not found in mitochondria in a human cell line [ 52 ].…”

The Effect of 2-Ketobutyrate on Mitochondrial Substrate-Level Phosphorylation

“…The decrease in NAD + provision to KGDHC with the aim of diminishing mSLP is the strategy followed by including β-hydroxybutyrate in the media (supporting NADH generation by β-hydroxybutyrate dehydrogenase) relying on the fact that KGDHC activity is important for mSLP [7]. Provision of NAD + to KGDHC in the absence of oxidative phosphorylation occurs through mitochondrially-localized diaphorases, [17] such as mitochondrially-localized NQO1 [18]. The effect of 2-KB inhibiting pyruvate oxidation has been published before [19], albeit this was only observed at very high 2-KB concentrations (20 mM).…”