Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

Abstract

In mitochondria, four respiratory-chain complexes drive oxidative phosphorylation by sustaining a proton-motive force across the inner membrane that is used to synthesize ATP. The question of how the densely packed proteins of the inner membrane are organized to optimize structure and function has returned to prominence with the characterization of respiratory-chain supercomplexes. Supercomplexes are increasingly accepted structural entities, but their functional and catalytic advantages are disputed. Notably, substrate "channeling" between the enzymes in supercomplexes has been proposed to confer a kinetic advantage, relative to the rate provided by a freely accessible, common substrate pool. Here, we focus on the mitochondrial ubiquinone/ubiquinol pool. We formulate and test three conceptually simple predictions of the behavior of the mammalian respiratory chain that depend on whether channeling in supercomplexes is kinetically important, and on whether the ubiquinone pool is partitioned between pathways. Our spectroscopic and kinetic experiments demonstrate how the metabolic pathways for NADH and succinate oxidation communicate and catalyze via a single, universally accessible ubiquinone/ubiquinol pool that is not partitioned or channeled. We reevaluate the major piece of contrary evidence from flux control analysis and find that the conclusion of substrate channeling arises from the particular behavior of a single inhibitor; we explain why different inhibitors behave differently and show that a robust flux control analysis provides no evidence for channeling. Finally, we discuss how the formation of respiratory-chain supercomplexes may confer alternative advantages on energy-converting membranes.

Keywords: mitochondria; respirasome; respiratory chain; supercomplex; ubiquinone.

Fig. 1.

Blue native PAGE analyses of the supercomplexes present in bovine heart mitochondria, membranes, and SMPs. (Left) Coomassie-stained gel showing that SMPs (prepared either with or without additional cyt c), membranes, and mitochondria all contain equivalent complements of supercomplexes. (Middle) Typical in-gel complex I activity assay (before and after incubation with NADH and NBT) showing the presence of complex I in supercomplexes. (Right) Typical in-gel complex II activity assay (before and after incubation with succinate, PMS, and NBT).

Fig. 2.

Spectral analysis of the redox status of the heme centers in complex III. (A) Individual difference spectra (relative to the starting spectrum) recorded following the addition of 5 mM succinate to 500 μg⋅mL⁻¹ SMPs in the presence of 5 μg⋅mL⁻¹ gramicidin; the SMPs were prepared with additional cyt c. The spectra are shown at the time points indicated (values are in seconds). (B) The fit between the difference spectrum recorded at 15 s and the modeled spectrum, calculated as a sum of the individual difference spectra shown in C and generated using the classical least squares method in matrix form. (C) Spectral components of the b_H, b_L, and c₁ hemes in complex III, cyt c, and complex IV from the model shown in B (the spectra of hemes a and a₃ in complex IV are combined and the background scatter omitted for simplicity). (D) Change in the peak difference absorbances (marked with asterisks in C) for the b_H, b_L, and c₁ hemes in complex III, and for cyt c, following addition of succinate. Conditions: 10 mM Tris-SO₄ (pH 7.5), 250 mM sucrose, 32 °C.

Fig. 3.

Reduction of complex III upon addition of NADH, succinate, or both to SMPs. The redox status of the heme centers in complex III and of cyt c in SMPs were followed spectroscopically, as described in Fig. 2. (A) Heme c₁; (B) cyt c; (C) heme b_L; (D) heme b_H. SMPs (500 μg⋅mL⁻¹, prepared with additional cyt c) were reduced anaerobically by addition of 5 mM succinate, 150 μM NADH, or 5 mM succinate and 150 μM NADH together in the presence of 5 μg⋅mL⁻¹ gramicidin. Three technical replicates of each experiment are presented as mean averages ± SEM (n = 3).

Fig. 4.

Comparison of the steady-state rates of individual and simultaneous NADH and succinate oxidation in SMPs and membranes. Light gray, contributions from succinate oxidation; dark gray, contributions from NADH oxidation. The dashed line above each dataset represents the combined individual rates. Values were deduced from simultaneous measurement of the rates of O₂ reduction and NADH oxidation (see Materials and Methods for details). Malonate (a competitive complex II inhibitor) was added to 0.145, 0.29, and 7.25 mM; when malonate was added the rates were too low to measure accurately in membranes without additional cyt c. Gramicidin (5 μg⋅mL⁻¹) was included in SMP experiments. Values reported are the mean average of at least three technical replicates and are ± SEM values.