bims-cytox1 2026-02-22 papers

Front Chem. 2025 ;13 1645343

The three-dimensional structure of a proton-pumping pathway, the H-pathway, is evolutionarily conserved in all three families of cytochrome c oxidase.

Atsuhiro Shimada, Tomitake Tsukihara, Shinya Yoshikawa.

The O2 reduction site of bovine cytochrome c oxidase (CcO) comprises two redox-active metal centers: Fe a3 and CuB. O2 is reduced at Fe a3 by four electrons transferred from cytochrome c in the P-side phase. Three proton-conducting pathways, D, K, and H, have been identified. Two distinct proton-pumping mechanisms, the D- and H-pathway mechanisms, proposed over 30 years ago, remain a subject of active debate. The former proposes that D-pathway transfers both pumping and water forming protons, whereas the latter proposes that the H-pathway transfers the pumping protons. CcOs are distributed across all aerobic organisms and are classified into evolutionarily related families: A, B, and C. In this study, we analyzed the common three-dimensional (3D) structural features of representative CcOs from each family to identify the proton-pumping system, assuming that the 3D structures responsible for the fundamental function of CcO, O2 reduction coupled with proton pumping, are evolutionarily conserved. Our analysis reveals that the 3D structural elements essential for proton pumping via the H-pathway mechanism are conserved across all three CcO families. These conserved elements include: 1) the site for loading and active release of pumping-protons to the P-side phase; 2) a water channel with a gate which opens to collect pumping protons from the N-side before the catalytic cycle starts and closes during the catalytic cycle to prevent leakage of pumping protons; 3) a water cluster located above the water-channel gate for storing pumping protons delivered from the water channel and transferring them in a timely manner to the proton-loading/release site when the gate is closed; and 4) a pumping-proton pool system, located below the water-channel gate, for the facile supply of protons to the water cluster when the gate opens. This structural conservation suggests that the H-pathway is responsible for proton pumping. Experimental results, supporting the D-pathway mechanism, do not disprove (are consistent with) the H-pathway mechanism. However, structural elements required to prevent pumping-proton leakage to the O2-reduction site and to the N-side surface, indispensable for the D-pathway as a proton pumping system, have not been identified experimentally.

Keywords: X-ray crystal structure; bioenergetics; conservativity of H-pathway; cytochrome c oxidase; heme-copper oxidase; proton-pump mechanism

DOI: https://doi.org/10.3389/fchem.2025.1645343

J Biol Chem. 2026 Feb 12. pii: S0021-9258(26)00148-1. [Epub ahead of print] 111278

Dynamic assessment of the allocation of copper to cytochrome c oxidase using size-exclusion chromatography (SEC) combined with inductively coupled plasma mass spectrometry (ICP-MS).

Dina Secic, Megan E Bischoff, Lucas Schmidt, Warunya Panmanee, Juechen Yang, Jarek Meller, Katherine E Vest, John T Cunningham, Julio A Landero, Maria F Czyzyk-Krzeska.

Copper (Cu) is an essential trace element required for mitochondrial respiration via its incorporation into cytochrome c oxidase (CuCOX), the terminal enzyme of the electron transport chain. Here, we employed size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), UV-Vis spectroscopy, and immunoblotting to identify and validate a high molecular weight Cu-containing peak in the SEC-ICP-MS chromatogram as representative of CuCOX activity. We demonstrate that this CuCOX peak is enhanced under metabolic conditions inducing oxidative phosphorylation, such as high Cu supplementation or galactose-containing media, and correlates with increased mitochondrial respiration. Using exogenous 63Cu tracing, we characterized the time- and dose-dependent incorporation of newly acquired Cu into CuCOX under elevated Cu conditions in renal cancer cells, modeling advanced clear cell renal cell carcinoma (ccRCC). RNA interference experiments targeting key Cu transporters revealed that CuCOX formation is independent of the high-affinity Cu importer CTR1, but instead relies on alternative transporters, including DMT1, LAT1, and the mitochondrial carrier SLC25A3, with transporter contributions dynamically reshaped during chronic adaptation to high Cu availability. In contrast, under standard low-Cu conditions, CTR1 remains required for cellular Cu uptake and CuCOX metallation. Together, these findings define context-dependent Cu trafficking pathways in renal cancer and establish SEC-ICP-MS as a sensitive platform for assessing CuCOX metallation and mitochondrial metabolism, with potential applications in biomarker discovery and therapeutic targeting in RCC.

Keywords: Copper; SEC-ICP-MS; cytochrome c oxidase; renal cancer

DOI: https://doi.org/10.1016/j.jbc.2026.111278

Biochemistry (Mosc). 2026 Jan;91(1): 178-187

Functional Divergence of bL36m Protein in Yeast and Human Mitochondrial Ribosomes.

Uliana E Piunova, Mariia V Baleva, Margarita S Lantsova, Ivan V Chicherin, Ruslan A Vasilev, Anastasia M Moysenovich, Sergey A Levitskii, Piotr A Kamenski.

L36 is a structural protein of the large ribosomal subunit of bacterial, mitochondrial, and chloroplast ribosomes. L36 stabilizes the peptidyl transferase center and the L7/L12 stalk, which is a binding site for the elongation factors during the translation cycle. According to the cryoelectron microscopy data, L36 incorporates into the large ribosomal subunit in both bacteria and mitochondria at the final assembly step. Bacterial L36 is not an essential protein, since deletion of its gene in bacteria did not impair the colony growth or reduce the mRNA translation levels. Deletion of the RTC6 gene coding for the mitochondrial homologue of L36 (bL36m) in Saccharomyces cerevisiae, impeded yeast growth on the media with non-fermentable carbon sources. Our findings indicate that the mitochondrial dysfunction associated with the absence of bL36m was caused by a decreased activity of cytochrome c oxidase complex that resulted from the selective disruption of synthesis of its subunits encoded in the mitochondrial genome. Furthermore, selective inhibition of mitochondrial protein synthesis did not induce critical structural abnormalities of mitochondrial ribosomes or reduce their ability to bind mRNA. Furthermore, we demonstrated that in contrast to S. cerevisiae, the absence of bL36m protein in human cells had no substantial impact on the synthesis of mitochondrially encoded proteins or mitochondrial ribosome assembly. However, the observed reduction in the mitochondrial respiration in the bL36m-deficient cells may be indicative of disturbances in the respiratory chain organization not associated with the mitochondrial translation.

Keywords: mitochondria; mitochondrial translation; regulation of translation

DOI: https://doi.org/10.1134/S000629792560348X