Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms

Abstract

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt-cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt-cyb encodes the core subunit (MT-CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt-cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human-associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt-cyb variations in key catalytic domains of MT-CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt-cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt-cyb-biochemical relationships in yeast provides a resource for future investigations in humans.

Keywords: MT-CYB; atovaquone; clomipramine; mitochondrial DNA; yeast model.

Figure 1

The catalytic core of yeast complex III. A: Redox active groups are located within three subunits that form the catalytic core: cytochrome c₁ (purple), the iron–sulfur protein/ISP (yellow), and cytochrome b (blue), which contains two b‐type hemes (b _l and b _h) and forms the two quinol binding sites: Q_o (site of quinol oxidation), with bound stigmatellin (gray) and Q_i (site of quinone reduction) with bound ubiquinone (green), located on opposite sides of the membrane. During catalytic turnover, a quinol molecule binds at the Q_o site is deprotonated, and transfers one electron through the [2Fe‐2S] cluster of the ISP and the c‐type heme of cytochrome c ₁ to cytochrome c. Following a bifurcated pathway, a second electron is transferred across the membrane to hemes b _l and _h and delivered to quinone bound at the Q_i site, forming a stable semiquinone. A second quinol oxidation event at the Q_o site completes the Q‐cycle with the formation of fully reduced quinol at the Q_i site. In the overall reaction, two molecules of quinol are oxidized to quinone at the Q_o site and one molecule of quinone is reduced to quinol at the Q_i site with the release of four protons to the positive side of the membrane and the uptake of two protons from the matrix. The figure was drawn using the coordinates 2IBZ of yeast complex III. B: Schematic presentation of cytochrome b and location of the residues of interest. The Q_i region is in green; the Q_o region is in gray. The helices are marked by letter (A–H). The positions of the residues (human numbering) studied here are shown by orange dots.

Figure 2

Location of the residues in yeast Q_o domain with bound atovaquone. Atovaquone (gray) is bound is a position similar to stigmatellin (Fig. 1A). Heme b _l is in red. Yeast numbering is used. Residues p.Gly143, p.Leu150, p.Thr127, p.Cys133, p.Cys134, p.Val135, p.Thr136, p.His141, and p.Ser172 are colored in yellow and their sidechains are shown. The figure was drawn using the coordinates 4PD4 of yeast complex III.

Figure 3

A: Atovaquone sensitivity of yeast mutant and wild‐type control complex III. IC₅₀s, midpoint inhibition concentrations, were estimated from the titration measurements and reported per complex III (Materials and Methods). Each measurement was repeated three times and averaged. Error bars represent standard deviations. B and C: Sensitivity of yeast mutant and wild‐type control respiratory growth to atovaquone. Atovaquone sensitivity is presented for each strain as the percentage of growth relative to wild‐type control, that is, untreated. Each measurement was repeated at least twice and averaged. Error bars represent standard deviations. B: Strain harboring single mutation p.Ser172Asp, Asn, Gly and p.Leu150Phe. C: Strain with multiple changes (HS) in the Q_o site that replace yeast residues by human equivalents.

Figure 4

A: Comparison of the yeast and mammalian Q_i sites. Superposition of yeast (blue) and bovine (pink) cytochrome b structures. Ubiquinol is in green and heme b _h, in red. The figure was drawn using the coordinates 4PD4 and 1PP9 of yeast and bovine complex III, respectively. The two structures were superposed using Chimera. Residues p.Ile17, p.Gly100, and p.Phe225 of yeast cytochrome b and p.Phe220, p.Gly101, and p.Phe220 of mammalian cytochrome b are shown. B: Possible interactions between p.Asn31 and helix E, and between p.Gly37Ser and ubiquinol. Cytochrome b is colored in blue, ubiquinol in green, p.Asn31 and p.Ser37 in yellow, waters as black sticks, and heme b _h in red. Possible hydrogen bonds are indicated in black lines. Minimum distance between p.Ser37 and ubiquinol p.Cys13 hydrogen is 1.0 Å. The structure was drawn using 2IBZ. In silico mutation p.Gly37Ser was introduced using Chimera.

Figure 5

Clomipramine sensitivity of yeast mutant and wild‐type control complex III. IC₅₀s, midpoint inhibition concentrations, were estimated from the titration measurements and normalized for complex III concentration (Materials and Methods). Each measurements was repeated three times and averaged. Error bars represent standard deviations. IFGD: p.Ile17Phe+p.Gly100Asp.

Figure 6

A: Complex III activity in wild‐type control and mutants. The ubiquinol cytochrome c reductase assays were performed as described in Materials and Methods. The activities measured at saturated substrate concentrations (40 mM decylubiquinol) were reported relative to complex III concentration. The measurements were repeated at least twice and averaged. The error bars represent standard deviations. B: Respiratory growth of wild‐type control and mutants. Serial dilutions in water of cells pregrown on glucose plates were spotted on plates containing either glucose (YPD, fermentative medium) or glycerol (YPG, respiratory medium) and incubated for two (YPD) to 4 days (YPG) at 28°C.