Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Abstract

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.

Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

“…Mitochondria can produce superoxide or H 2 O 2 from at least 10 different sites (16,23). Which sites are the major producers in vivo is unknown.…”

“…1. In order of their maximum capacities, they are as follows: III Qo 4 in complex III (17); I Q (18,19) and II F (20) in complexes I and II; O F , P F , and B F in the 2-oxoglutarate, pyruvate, and branched-chain 2-oxoacid dehydrogenase complexes (16); G Q in mitochondrial glycerol phosphate dehydrogenase (21); I F in complex I (16); E F in ETF/ETF:Q oxidoreductase (22); and D Q in dihydroorotate dehydrogenase (23).…”

Mitochondrial ROS metabolism: 10 Years later

“…Recently, Brand and coworkers [54] separated two components of ROS generation linked to DHO oxidation. In skeletal muscle mitochondria, where the level and activity of the enzyme are so low that DHO-dependent oxygen consumption could not be detected, they were able to detect measurable DHO-dependent H 2 O 2 production.…”

“…In skeletal muscle mitochondria, where the level and activity of the enzyme are so low that DHO-dependent oxygen consumption could not be detected, they were able to detect measurable DHO-dependent H 2 O 2 production. Based on sensitivity to specific inhibitors malonate and atpenin A5, it was concluded that the majority of ROS apparently originated from CII [54]. The rate of superoxide/H 2 O 2 generation attributed to DHO DH itself (~20 pmol/min per mg protein) constituted less than 10% of the total rate.…”

“…The rate of superoxide/H 2 O 2 generation attributed to DHO DH itself (~20 pmol/min per mg protein) constituted less than 10% of the total rate. Based on the inhibitory effect of brequinar that blocks electron transfer between the flavin and ubiquinone-binding site of DHO DH, the authors concluded that the site of ROS generation is the ubiquinone-binding site [54]. However, brequinar has been shown to block normal dehydrogenase activity (electron transfer from DHO to coenzyme Q) but not the ROS producing “DHO oxidase” activity of isolated enzyme [52], rendering this conclusion controversial.…”