A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

Abstract

Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone‐10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self‐assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady‐state catalysis. The system was used to measure a fully‐defined K M value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.

Energy conversion, redox catalysis and generation of reactive oxygen species by respiratory complex I

“…Kinetic studies using B. taurus complex I have revealed that the apparent second order rate constant for NADH binding (represented by k cat NADH /K M NADH , where k cat NADH is the (maximum) rate of NADH oxidation observed at saturating NADH concentration and K M is the Michaelis constant, equivalent to the NADH concentration required for half the maximum rate) is ~ 7.5 × 10 7 M − 1 s − 1 , approaching the diffusion-controlled limit, and that k cat NADH (which includes both reversible hydride transfer and NAD + dissociation) is greater than 15,000 s − 1 [14] , [19] . In comparison, the maximum rate of NADH:ubiquinone oxidation that has been observed is ~ 400 s − 1 [20] , [21] .…”

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…Compared with all the membrane mimetic systems explored in this review liposomes offer several unique advantages. First, the large size of liposomes allows for multi component systems (proteins, ligands and substrates) to be reconstituted [ 259 ]. Furthermore, the aqueous lumen can encapsulate soluble components allowing entire GPCR signalling pathways to be reconstituted, for example the M1 muscarinic receptor along with G q/11 βγ and phospholipase C in liposomes were induced to produce inositol triphosphate upon exposure of carbachol [ 260 ].…”

Using a chimeric respiratory chain and EPR spectroscopy to determine the origin of semiquinone species previously assigned to mitochondrial complex I

“…The addition of AOX provides a rapid secondary quinol oxidation route ( Fig. 1), such that NADH:O 2 turnover is capable of exceeding turnover by the canonical respiratory chain in an AOX-concentration dependent manner [24,25]. Thus, to prevent the substrate from becoming exhausted and ensure turnover during EPR sample preparation, AOX was titrated to a low level, at which the flux through the complex I-AOX pathway matched that through the canonical chain (Additional file 1, Figure S1).…”

“…The alternative oxidase (AOX) from Trypanosoma brucei brucei was prepared and incorporated into SMP membranes as described previously [24,25]. A 100-μL sample of SMPs was incubated with the desired quantity of AOX (from a 3-5 mg/mL stock) on ice for at least 30 min.…”

“…Although a chimeric respiratory chain using SMPs and the purified cytochrome bd complex from Escherichia coli has been described previously [21], it is unsuitable for SQ studies owing to the very stable and therefore dominant cytochrome bd SQ [22,23]. Addition of the alternative oxidase (AOX) from Trypanosoma brucei brucei to SMPs creates 'AOX-SMPs', containing a quinone-cycling system between complex I and AOX and with NADH:O 2 turnover sensitive to complex I inhibitors (such as piericidin A and rotenone) and the AOX inhibitor ascofuranone [24,25]. As no SQ intermediates have been detected by EPR spectroscopy in wild-type AOX [26,27], incorporation of this terminal oxidase into SMPs represents an intriguing new platform to investigate the SQs that are generated during sustained NADH oxidation by complex I.…”