Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

Abstract

Mitochondria are the powerhouses of eukaryotic cells as they feed metabolism with its major substrate. Oxidative-phosphorylation relies on the generation, by an electron/proton transfer chain, of an electrochemical transmembrane potential utilized to synthesize ATP. Although these fundamental principles are not a matter of debate, the emerging picture of the respiratory chain diverges from the linear and fluid scheme. Indeed, a growing number of pieces of evidence point to membrane compartments that possibly restrict the diffusion of electron carriers, and to supramolecular assembly of various complexes within various kinds of supercomplexes that modulate the thermodynamic and kinetic properties of the components of the chain. Here, we describe a method that allows the unprecedented time-resolved study of the respiratory chain in intact cells that is aimed at assessing these hypotheses. We show that, in yeast, cytochrome c is not trapped within supercomplexes and encounters no particular restriction to its diffusion which questions the functional relevance of these supramolecular edifices.

Fig. 1.

Photolysis of CO in whole cells. (A) Spectrum of CO photolysis from heme a₃ in a suspension of yeast (W303) cells (open symbols) under a pure CO atmosphere. A spectrum from the literature (32), red-shifted by two nanometers, is shown for comparison (solid line). (B) Kinetics of CO rebinding to heme a₃ after photolysis, at P_CO = 1 atm (closed symbols) and P_CO = 0.5 atm/P_Ar = 0.5 atm (open symbols), and monoexponential fits (solid lines). Plots and linear fits of the apparent rate constants against [CO] are shown in the inset.

Fig. 2.

Monitoring by visible spectrophotometry the photoactivated reactions of the respiratory chain with oxygen. Spectra of whole yeast (W303) cells at various times after CO photolysis, under an atmosphere of P_CO = 0.94 and P_O2 = 0.06. The excitation light was switched from 605 to 430 nm in order to explore the red part of the spectrum (see Materials and Methods). Notable spectrophotometric signals include: CO photolysis from CcOx (see Fig. 1A), oxidation of CcOx (troughs at 445 nm and 605 nm), oxidation of cytochrome c (troughs at 420 nm, 520 nm, and 550 nm), oxidation of flavoproteins (band in the 440–500 nm region), and oxidation of b-type hemes (slight troughs at 430 nm and 560 nm, seen at longer times).

Fig. 3.

Oxidation kinetics of CcOx under various oxygen concentrations. (A) Kinetics of CcOx oxidation in the W303 strain at different [O₂], monitored at 445 nm, with correction for contributions from flavoproteins and cytochrome c (see Materials and Methods). Solid lines in (A and B) are biexponential fits of the deconvoluted kinetics in the W303 strain. (B) Kinetics of CcOx oxidation in the BY strain at various [O2]. (C) Plots and linear fits of the apparent rate constants of the fast phase of CcOx oxidation against [O₂] for the W303 (orange symbols) and BY (blue symbols) strains.

Fig. 4.

Kinetics of cytochrome c oxidation at different [O₂], monitored at 551 nm. (A) W303 strain. (B) BY strain. (C) kinetics for [O2] = 156 μM of cytochrome c oxidation in the W303 strain, with monoexponential (gray line) and biexponential fits (orange line) of the data; the corresponding residuals are shown below.

Fig. 5.

Progressive oxidation of cytochrome c by a series of laser flashes after inhibition of cytochrome bc₁. Relative amount of cytochrome c oxidized on a series of flashes at different laser intensities (Φ_CO), monitored at 470 nm (see Materials and Methods) in the W303 strain. Gray dashed lines are fits of the data with a geometrical probability law, constraining the value of α = 0.08 (see text).

Fig. P1.

Mitochondrial respiration involves electron transfer from NADH to molecular oxygen. This overall reaction is mediated by membrane-embedded proteins (here depicted as complex I, complex III, and complex IV); quinones (in green), which are membrane soluble and transfer electrons between complexes I and III; and cytochrome c, which is soluble in the intermembrane space and transfers electrons between complexes III and IV. In A is shown the “liquid model” in which soluble electron carriers freely diffuse between the membrane-embedded complexes. In B are shown “respirasomes” or the solid-state model, in which the membrane complexes are assembled within supramolecular edifices that trap the soluble electron carriers and thereby restrict their diffusion. In C is shown the compartmented model, in which the intricate folding of the inner membrane defines local compartments that allow the free diffusion of cytochrome c but prevent equilibration between compartments.