A mitochondrial protein-folding pathway has been linked to heme production and cellular respiration, revealing a mechanism that may help explain how inherited mutations in augmenter of liver regeneration (ALR) disrupt cellular energy supply.
ALR is a sulfhydryl oxidase best known for maintaining the mitochondrial disulfide relay. In that pathway, ALR reoxidizes MIA40, which introduces stabilizing disulfide bonds into proteins entering the mitochondrial intermembrane space. Researchers at the University of Cologne have now shown that ALR also stabilizes coproporphyrinogen III oxidase (CPOX), a heme-biosynthesis enzyme.
To identify ALR interaction partners, the team trapped transient mixed disulfide intermediates formed during oxidative protein folding. Tagged ALR was enriched under native and denaturing conditions, then analyzed by quantitative mass spectrometry, revealing several ALR-associated mitochondrial proteins, including CPOX.
Follow-up immunoprecipitation, redox-shift, cysteine-mutant, and in vitro oxidation assays showed that ALR introduces a disulfide bond into CPOX during biogenesis. Without the relevant cysteine residues, CPOX retained enzymatic activity in vitro but became unstable inside the mitochondrial intermembrane space.
“Our findings show that oxidative protein folding in mitochondria plays a far greater role than previously thought,” said lead author Julia Racho in the University of Cologne press release. “Not only does it ensure that proteins adopt their correct shape, but it also directly supports a vital metabolic process.”
The team then tested the metabolic consequences using CPOX knockout cells and rescue experiments with wild-type or disulfide-deficient CPOX variants. High-performance liquid chromatography-mass spectrometry showed disrupted porphyrin homeostasis when CPOX was absent or destabilized, including reduced heme production and accumulation of upstream heme-biosynthesis intermediates. Cells lacking functional CPOX also showed impaired growth under conditions requiring mitochondrial respiration.
The researchers further showed that relocating disulfide-deficient CPOX to the cytosol partially bypassed its instability in the mitochondrial intermembrane space. But this spatial workaround made heme biosynthesis less efficient and led to accumulation of protoporphyrinogen IX, a redox-active intermediate that sensitized cells to death under oxidative stress.
By directly stabilizing CPOX, ALR connects mitochondrial protein folding to heme biosynthesis, linking its redox role to a pathway required for respiration. The authors now plan to test whether other mitochondrial enzymes depend on similar stabilization, a question that could also clarify how ALR mutations disrupt energy supply in inherited mitochondrial disorders.
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