bims-cytox1 2023-05-28 papers

Acta Pharmacol Sin. 2023 May 22.

COX17 restricts renal fibrosis development by maintaining mitochondrial copper homeostasis and restoring complex IV activity.

Sai-Ya Zhu, Wen-Qian Zhou, Yang-Yang Niu, Chao Zheng, Xi Liu, Ying-Ying Zhang, Chen Yu.

Renal fibrosis relies on multiple proteins and cofactors in its gradual development. Copper is a cofactor of many enzymes involved in renal microenvironment homeostasis. We previously reported that intracellular copper imbalance occurred during renal fibrosis development and was correlated with fibrosis intensity. In this study, we investigated the molecular mechanisms of how copper affected renal fibrosis development. Unilateral ureteral obstruction (UUO) mice were used for in vivo study; rat renal tubular epithelial cells (NRK-52E) treated with TGF-β1 were adapted as an in vitro fibrotic model. We revealed that the accumulation of copper in mitochondria, rather than cytosol, was responsible for mitochondrial dysfunction, cell apoptosis and renal fibrosis in both in vivo and in vitro fibrotic models. Furthermore, we showed that mitochondrial copper overload directly disrupted the activity of respiratory chain complex IV (cytochrome c oxidase), but not complex I, II and III, which hampered respiratory chain and disrupted mitochondrial functions, eventually leading to fibrosis development. Meanwhile, we showed that COX17, the copper chaperone protein, was significantly upregulated in the mitochondria of fibrotic kidneys and NRK-52E cells. Knockdown of COX17 aggravated mitochondrial copper accumulation, inhibited complex IV activity, augmented mitochondrial dysfunction and led to cell apoptosis and renal fibrosis, whereas overexpression of COX17 could discharge copper from mitochondria and protect mitochondrial function, alleviating renal fibrosis. In conclusion, copper accumulation in mitochondria blocks complex IV activity and induces mitochondrial dysfunction. COX17 plays a pivotal role in maintaining mitochondrial copper homeostasis, restoring complex IV activity, and ameliorating renal fibrosis.

Keywords: COX17; NRK-52E cells; copper; cytochrome c oxidase; mitochondria; renal fibrosis

DOI: https://doi.org/10.1038/s41401-023-01098-3

bioRxiv. 2023 May 10. pii: 2023.05.08.539888. [Epub ahead of print]

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Izumi Ishigami, Sergio Carbajo, Nadia Zatsepin, Masahide Hikita, Chelsie E Conrad, Garrett Nelson, Jesse Coe, Shibom Basu, Thomas Grant, Matthew H Seaberg, Raymond G Sierra, Mark S Hunter, Petra Fromme, Raimund Fromme, Denis L Rousseau, Syun-Ru Yeh.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu B ) and a heme a 3 iron, where O 2 binds and is reduced to water. CO is a versatile O 2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (Cu B -CO), where CO is bound to Cu B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the Cu B and the heme a 3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

DOI: https://doi.org/10.1101/2023.05.08.539888

Redox Biol. 2023 May 15. pii: S2213-2317(23)00141-6. [Epub ahead of print]63 102740

The metabolic state of the heart regulates mitochondrial supercomplex abundance in mice.

Yuting Zheng, Andrew A Gibb, Hongkai Xu, Siqi Liu, Bradford G Hill.

Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O2. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (GlycoLo), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (GlycoHi), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.

Keywords: Glycolysis; Heart; Metabolism; Mitochondria; Respirasome; Supercomplex

DOI: https://doi.org/10.1016/j.redox.2023.102740

Front Chem. 2023 ;11 1108190

Recent progress in experimental studies on the catalytic mechanism of cytochrome c oxidase.

Atsuhiro Shimada, Tomitake Tsukihara, Shinya Yoshikawa.

Cytochrome c oxidase (CcO) reduces molecular oxygen (O2) to water, coupled with a proton pump from the N-side to the P-side, by receiving four electrons sequentially from the P-side to the O2-reduction site-including Fea3 and CuB-via the two low potential metal sites; CuA and Fea. The catalytic cycle includes six intermediates as follows, R (Fea3 2+, CuB 1+, Tyr244OH), A (Fea3 2+-O2, CuB 1+, Tyr244OH), Pm (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244O•), F (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244OH), O (Fea3 3+-OH-, CuB 2+-OH-, Tyr244OH), and E (Fea3 3+-OH-, CuB 1+-H2O, Tyr244OH). CcO has three proton conducting pathways, D, K, and H. The D and K pathways connect the N-side surface with the O2-reduction site, while the H-pathway is located across the protein from the N-side to the P-side. The proton pump is driven by electrostatic interactions between the protons to be pumped and the net positive charges created during the O2 reduction. Two different proton pump proposals, each including either the D-pathway or H-pathway as the proton pumping site, were proposed approximately 30 years ago and continue to be under serious debate. In our view, the progress in understanding the reaction mechanism of CcO has been critically rate-limited by the resolution of its X-ray crystallographic structure. The improvement of the resolutions of the oxidized/reduced bovine CcO up to 1.5/1.6 Å resolution in 2016 provided a breakthrough in the understanding of the reaction mechanism of CcO. In this review, experimental studies on the reaction mechanism of CcO before the appearance of the 1.5/1.6 Å resolution X-ray structures are summarized as a background description. Following the summary, we will review the recent (since 2016) experimental findings which have significantly improved our understanding of the reaction mechanism of CcO including: 1) redox coupled structural changes of bovine CcO; 2) X-ray structures of all six intermediates; 3) spectroscopic findings on the intermediate species including the Tyr244 radical in the Pm form, a peroxide-bound form between the A and Pm forms, and Fr, a one-electron reduced F-form; 4) time resolved X-ray structural changes during the photolysis of CO-bound fully reduced CcO using XFEL; 5) a simulation analysis for the Pm→Pr→F transition.

Keywords: O2 reduction mechanism; X-ray crystal structure; bioenergetics; cytochrome c oxidase; enzyme reaction mechanism; proton-pump mechanism

DOI: https://doi.org/10.3389/fchem.2023.1108190