Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Escherichia coli fumarase (FumC) and oxaloacetate-decarboxylating malate dehydrogenase (MaeB) were overexpressed in BL21(DE3) cells transformed with the maeB or fumC plasmid, exactly as previously described (Bologna et al., 2007, Jones and Hirst, 2013, Weaver et al., 1995). Cultures were grown at 32°C in LB medium supplemented with 50 μg ml-1 ampicillin and induced at OD600 ∼0.6 with IPTG (1 mM for FumC and 0.1 mM for MaeB) for 18 h at 20°C. Cells were harvested by centrifugation (3 500 g, 10 min, 4°C), resuspended in buffer A (50 mM potassium phosphate [pH 7.8 at 4°C] and 300 mM NaCl) for FumC or buffer B (20 mM Tris-HCl [pH 7.4 at 4°C], 100 mM NaCl, 25 mM imidazole, and 10% (w/v) glycerol) for MaeB, then lysed using a Constant cell disruption system (Constant Systems Ltd) at 30 kPa. The lysates were centrifuged (150 000 g, 45 min, 4°C) then the supernatants were loaded onto pre-equilibrated 25 ml Ni-Sepharose 6 Fast Flow columns (GE Healthcare). The columns were washed using the respective buffers supplemented with 60 mM imidazole. FumC and MaeB were eluted with the appropriate buffer supplemented with 400 mM or 300 mM imidazole, respectively. Fractions were pooled, concentrated then dialyzed overnight in 10 mM Tris-SO4 [pH 7.0 at 4°C], 5 mM EDTA, and 1 mM dithiothreitol, for FumC and 60 mM Tris-SO4 [pH 7.4 at 4°C], 20 mM β-mercaptoethanol, 25 mM imidazole, and 10% (w/v) glycerol for MaeB.

Protein concentrations were determined by the bicinchoninic acid assay, following solubilization of SMPs with 2% SDS.

All assays were conducted in a standard assay buffer (10 mM Tris-SO4 [pH 7.5 at 32°C] and 250 mM sucrose) at 32°C either in 1 mL cuvettes or in 96-well plates using a Molecular Devices SpectraMax Plus 384 microplatereader. NADH:O2 oxidoreduction was measured spectroscopically in 200 μM NADH (ɛ = 4.81 mM-1 cm-1 at 340 − 380 nm) (Birrell et al., 2009). Titration curves were measured in 96-well plates then the maximum rate for each curve was confirmed in cuvette experiments. Succinate:O2 oxidoreduction was determined indirectly by monitoring the NADPH (ɛ = 4.81 mM-1 cm-1 at 340 − 380 nm) generated by a coupled enzyme assay system composed of 5 mM succinate, 2 mM NADP+, 2 mM MgSO4, 1 mM K2SO4, 60 μg mL-1 FumC and 300 μg mL-1 MaeB (Jones and Hirst, 2013).

SMP samples were solubilized on ice at 2.5 mg mL-1, using 1% digitonin (high purity, Calbiochem) and 1x NativePAGE sample buffer (Invitrogen) for 20 min. Protein concentrations were determined following centrifugation at 14 000 g for 30 min at 4°C, then the samples were aliquoted and flash frozen for storage at −80°C. 10 μg of solubilized material was applied in each lane of 3-12% Bis-Tris NativePAGE gels (Invitrogen), run according to the manufacturer’s instructions. Proteins were visualized using Coomassie R250 staining or by an in-gel NADH oxidase activity assay using a 20 mM Tris-HCl [pH 7.0 at 23°C] solution of 100 μM NADH and 0.5 mg mL−1 nitrotetrazolium blue (Wittig et al., 2007).

Assays were conducted in an anaerobic glovebox at 32°C in assay buffer containing 1 mM NAD+, 10 mM succinate, 1 mM MgATP and 50 μg mL-1 SMPs. The MgATP was added to the SMPs before the other substrates and the NADH concentration monitored as described above. Δp titrations were carried out using 100 μM NADH, 1 mM NAD+, 500 μM succinate, 1 mM MgATP and 50 μg mL-1 SMPs (prepared without additional cytochrome c). The concentration of fumarate was varied from 25 μM to 40 mM to set ΔE according to Equation 1 with ΔEm,7.5 = 0.33 V (Nicholls and Ferguson, 2013). Gramicidin (5 μg mL-1) was added from a DMSO stock solution so that the DMSO concentration did not exceed 0.5% (v/v). Δp was determined from −ΔE/2 at the x-intercept of the data curve, as per Equation 2.

The CI stoichiometry was determined using a method based on that of Jones and coworkers (Jones et al., 2017). SMPs supplemented with 0.02 mg AOX mg-1 were used to give appropriate rates of NADH oxidation and ATP synthesis. Ascofuranone (0.5 μM) and NaCN (0.4 mM) were used to inhibit the AOX or CIII/CIV pathways, respectively. ADP-ribose at 4, 6, 8 and 10 mM was used to give a range of NADH oxidation rates (Zharova and Vinogradov, 1997) for the CI/CIII/CIV pathway, and 0, 0.25, 0.5 and 1 mM ADP-ribose were used for the CI/AOX pathway. Linear regression through the origin of the rates of ATP synthesis versus the rates of NADH oxidation allowed for comparison of the slopes and calculation of the CI H+/2e– stoichiometry. ATP concentrations were measured using the Roche ATP Bioluminescence assay kit CLS II in white 96-well plates using a Molecular Devices SpectraMax Gemini XPS microplate reader by comparison to known ATP/ADP standard solutions.

The number of replicates, mean and standard deviation of measurements are reported in the relevant figure legends. Linear regressions and two-tailed t-tests were carried out using GraphPad Prism 7.

We thank Andrew Jones (MBU) for helpful suggestions; C. Humphreys & Sons Abattoir, Chelmsford, and Shane Palmer (MBU) for assistance with enzyme sample preparation; and Kiyoshi Kita (Nagasaki University) for ascofuranone. This work was supported by the Medical Research Council (grant numbers MC_U105663141 and MC_UU_00015/2).

J.G.F. conducted the experiments; J.H. and J.G.F. designed experiments and wrote the paper.