Mutations in the accessory subunit NDUFB10 result in isolated complex I deficiency and illustrate the critical role of intermembrane space import for complex I holoenzyme assembly.

An infant presented with fatal infantile lactic acidosis and cardiomyopathy, and was found to have profoundly decreased activity of respiratory chain complex I in muscle, heart and liver. Exome sequencing revealed compound heterozygous mutations in NDUFB10, which encodes an accessory subunit located within the PD part of complex I. One mutation resulted in a premature stop codon and absent protein, while the second mutation replaced the highly conserved cysteine 107 with a serine residue. Protein expression of NDUFB10 was decreased in muscle and heart, and less so in the liver and fibroblasts, resulting in the perturbed assembly of the holoenzyme at the 830 kDa stage. NDUFB10 was identified together with three other complex I subunits as a substrate of the intermembrane space oxidoreductase CHCHD4 (also known as Mia40). We found that during its mitochondrial import and maturation NDUFB10 transiently interacts with CHCHD4 and acquires disulfide bonds. The mutation of cysteine residue 107 in NDUFB10 impaired oxidation and efficient mitochondrial accumulation of the protein and resulted in degradation of non-imported precursors. Our findings indicate that mutations in NDUFB10 are a novel cause of complex I deficiency associated with a late stage assembly defect and emphasize the role of intermembrane space proteins for the efficient assembly of complex I.

Mitochondrial disulfide relay and its substrates: mechanisms in health and disease.

Eukaryotic cells harbor membrane-enclosed compartments to spatially separate different biochemical processes. As a result, proteins that become synthesized in the cytosol but fulfill their function in another compartment require translocation machineries. In the intermembrane space (IMS) of mitochondria, the mitochondrial disulfide relay is responsible for the import of many soluble proteins in an oxidation-dependent manner. These IMS proteins carry out important tasks and therefore their import, folding and maintenance are crucial for the remainder of the cell. In this review, we first describe the machinery for oxidative protein folding in the IMS and then focus on recent developments, which especially concern the mammalian machinery, its substrates and its physiological role.

Erv1 and Cytochrome c Mediate Rapid Electron Transfer via A Collision-Type Interaction.

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O2), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.

Vectorial Import via a Metastable Disulfide-Linked Complex Allows for a Quality Control Step and Import by the Mitochondrial Disulfide Relay.

Disulfide formation in the mitochondrial intermembrane space (IMS) is an essential process. It is catalyzed by the disulfide relay machinery, which couples substrate import and oxidation. The machinery relies on the oxidoreductase and chaperone CHCHD4-Mia40. Here, we report on the driving force for IMS import and on a redox quality control mechanism. We demonstrate that unfolded reduced proteins, upon translocation into the IMS, initiate formation of a metastable disulfide-linked complex with CHCHD4. If this interaction does not result in productive oxidation, then substrates are released to the cytosol and degraded by the proteasome. Based on these data, we propose a redox quality control step at the level of the disulfide-linked intermediate that relies on the vectorial nature of IMS import. Our findings also provide the mechanistic framework to explain failures in import of numerous human disease mutants in CHCHD4 substrates.

The mitochondrial oxidoreductase CHCHD4 is present in a semi-oxidized state in vivo.

Disulfide formation in the mitochondrial intermembrane space is an essential process catalyzed by a disulfide relay machinery. In mammalian cells, the key enzyme in this machinery is the oxidoreductase CHCHD4/Mia40. Here, we determined the in vivo CHCHD4 redox state, which is the major determinant of its cellular activity. We found that under basal conditions, endogenous CHCHD4 redox state in cultured cells and mouse tissues was predominantly oxidized, however, degrees of oxidation in different tissues varied from 70% to 90% oxidized. To test whether differences in the ratio between CHCHD4 and ALR might explain tissue-specific differences in the CHCHD4 redox state, we determined the molar ratio of both proteins in different mouse tissues. Surprisingly, ALR is superstoichiometric over CHCHD4 in most tissues. However, the levels of CHCHD4 and the ratio of ALR over CHCHD4 appear to correlate only weakly with the redox state, and although ALR is present in superstoichiometric amounts, it does not lead to fully oxidized CHCHD4.