Resonance Raman spectral analysis of the heme site structure of cytochrome c oxidase with its positive regulator CHCHD2.

Cytochrome c oxidase (CcO) reduces O2, pumps protons in the mitochondrial respiratory chain, and is essential for oxygen consumption in the cell. The coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2; also known as mitochondrial nuclear retrograde regulator 1 [MNRR1], Parkinson's disease 22 [PARK22] and aging-associated gene 10 protein [AAG10]) is a protein that binds to CcO from the intermembrane space and positively regulates the activity of CcO. Despite the importance of CHCHD2 in mitochondrial function, the mechanism of action of CHCHD2 and structural information regarding its binding to CcO remain unknown. Here, we utilized visible resonance Raman spectroscopy to investigate the structural changes around the hemes in CcO in the reduced and CO-bound states upon CHCHD2 binding. We found that CHCHD2 has a significant impact on the structure of CcO in the reduced state. Mapping of the heme peripheries that result in Raman spectral changes in the structure of CcO highlighted helices IX and X near the hemes as sites where CHCHD2 takes action. Part of helix IX is exposed in the intermembrane space, whereas helix X, located between both hemes, may play a key role in proton uptake to a proton-loading site in the reduced state for proton pumping. Taken together, our results suggested that CHCHD2 binds near helix IX and induces a structural change in helix X, accelerating proton uptake.

Regulation of COX Assembly and Function by Twin CX9C Proteins-Implications for Human Disease.

Oxidative phosphorylation is a tightly regulated process in mammals that takes place in and across the inner mitochondrial membrane and consists of the electron transport chain and ATP synthase. Complex IV, or cytochrome c oxidase (COX), is the terminal enzyme of the electron transport chain, responsible for accepting electrons from cytochrome c, pumping protons to contribute to the gradient utilized by ATP synthase to produce ATP, and reducing oxygen to water. As such, COX is tightly regulated through numerous mechanisms including protein-protein interactions. The twin CX9C family of proteins has recently been shown to be involved in COX regulation by assisting with complex assembly, biogenesis, and activity. The twin CX9C motif allows for the import of these proteins into the intermembrane space of the mitochondria using the redox import machinery of Mia40/CHCHD4. Studies have shown that knockdown of the proteins discussed in this review results in decreased or completely deficient aerobic respiration in experimental models ranging from yeast to human cells, as the proteins are conserved across species. This article highlights and discusses the importance of COX regulation by twin CX9C proteins in the mitochondria via COX assembly and control of its activity through protein-protein interactions, which is further modulated by cell signaling pathways. Interestingly, select members of the CX9C protein family, including MNRR1 and CHCHD10, show a novel feature in that they not only localize to the mitochondria but also to the nucleus, where they mediate oxygen- and stress-induced transcriptional regulation, opening a new view of mitochondrial-nuclear crosstalk and its involvement in human disease.

Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) rescues the cellular phenotype of MELAS by inducing homeostatic mechanisms.

MNRR1 (CHCHD2) is a bi-organellar regulator of mitochondrial function that directly activates cytochrome c oxidase in the mitochondria and functions in the nucleus as a transcriptional activator for hundreds of genes. Since MNRR1 depletion contains features of a mitochondrial disease phenotype, we evaluated the effects of forced expression of MNRR1 on the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) syndrome. MELAS is a multisystem encephalomyopathy disorder that can result from a heteroplasmic mutation in the mitochondrial DNA (mtDNA; m.3243A > G) at heteroplasmy levels of ∼50 to 90%. Since cybrid cell lines with 73% m.3243A > G heteroplasmy (DW7) display a significant reduction in MNRR1 levels compared to the wild type (0% heteroplasmy) (CL9), we evaluated the effects of MNRR1 levels on mitochondrial functioning. Overexpression of MNRR1 in DW7 cells induces the mitochondrial unfolded protein response (UPRmt), autophagy, and mitochondrial biogenesis, thereby rescuing the mitochondrial phenotype. It does so primarily as a transcription activator, revealing this function to be a potential therapeutic target. The role of MNRR1 in stimulating UPRmt, which is blunted in MELAS cells, was surprising and further investigation uncovered that under conditions of stress the import of MNRR1 into the mitochondria was blocked, allowing the protein to accumulate in the nucleus to enhance its transcription function. In the mammalian system, ATF5, has been identified as a mediator of UPRmt MNRR1 knockout cells display an ∼40% reduction in the protein levels of ATF5, suggesting that MNRR1 plays an important role upstream of this known mediator of UPRmt.

MNRR1, a Biorganellar Regulator of Mitochondria.

The central role of energy metabolism in cellular activities is becoming widely recognized. However, there are many gaps in our knowledge of the mechanisms by which mitochondria evaluate their status and call upon the nucleus to make adjustments. Recently, a protein family consisting of twin CX9C proteins has been shown to play a role in human pathophysiology. We focus here on two family members, the isoforms CHCHD2 (renamed MNRR1) and CHCHD10. The better studied isoform, MNRR1, has the unusual property of functioning in both the mitochondria and the nucleus and of having a different function in each. In the mitochondria, it functions by binding to cytochrome c oxidase (COX), which stimulates respiration. Its binding to COX is promoted by tyrosine-99 phosphorylation, carried out by ABL2 kinase (ARG). In the nucleus, MNRR1 binds to a novel promoter element in COX4I2 and itself, increasing transcription at 4% oxygen. We discuss mutations in both MNRR1 and CHCHD10 found in a number of chronic, mostly neurodegenerative, diseases. Finally, we propose a model of a graded response to hypoxic and oxidative stresses, mediated under different oxygen tensions by CHCHD10, MNRR1, and HIF1, which operate at intermediate and very low oxygen concentrations, respectively.