Biology subjects

Kovalova, T.

Publications and source records attributed to Kovalova, T..

3 recordsLinked to original sources

bioRxiv2026

A Carboxylate Switch Point Controls Long-Range Energy Transduction in Respiratory Complex I

Complex I is a highly intricate membrane-bound protein that powers the cellular energy metabolism by a long-range (>300 [A]) proton-coupled electron transfer (PCET) reaction. Here, we investigate the unknown coupling mechanism of Complex I by probing the charge transfer reaction along its functionally central carboxylate pathway (E-channel). By combining biophysical and site-directed mutagenesis experiments with high-resolution (2.6-2.7 [A]) cryo-electron microscopy (cryo-EM) and multiscale simulations, we identify a conserved carboxylate switch point (D79NuoA) that mediates proton transfer by establishing a kinetic gate that couples the redox chemistry to proton pumping. We find that mutation of the identified site, as found in patients suffering from severe neurodegenerative disorders, perturbs the charge transfer mechanism, and results in a drastic (>80%) reduction of the long-range PCET activity. Our combined findings illustrate mechanistic principles of molecular gates underlying long-range charge transfer reactions, and show how disease mutations perturb the function of conserved switch points in energy transduction.

biophysics↗

bioRxiv2025

The Mycobacterium smegmatis bd-II terminal oxidase employs a 1 carboxylate shift mechanism

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here we characterise the first qOR-2 type bd oxidase, the cyt bd-II from Mycobacterium smegmatis, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. By over-expressing the appCB operon in its native host, we produce a highly active bd-II (kcat=30 e-s-1) that together with a high-resolution (2.8 [A]) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that O2-scavenging activates a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues that regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

biochemistry↗

bioRxiv2021

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic Gram-positive bacterium belonging to the Actinobacteria phylum, which also includes the pathogen Mycobacterium tuberculosis. In the respiratory chain of these bacteria, complexes III (CIII) and IV (CIV) form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. Electron transfer within the CIII2CIV2 supercomplex is linked to transmembrane proton translocation, which maintains an electrochemical proton gradient that drives ATP synthesis and transport processes. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 [A] resolution. The structure shows a central CIII2 dimer flanked by a CIV on each side. One menaquinone is bound in each of the QN and QP sites in each CIII, near the cytoplasmic and periplasmic sides, respectively. In addition, we identified a menaquinone positioned ~14 [A] from heme bL on the periplasmic side. A di-heme cyt. cc subunit provides an electronic connection between each CIII monomer and the adjacent CIV. In CIII2, the Rieske iron-sulfur (FeS) proteins are positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a novel path that conducts protons into CIV.

biochemistry↗