Cytochrome c Oxidase Detection in Human Fibroblasts Using Scanning Electrochemical Microscopy

Abstract

Cytochrome C Oxidase Deficiency (COXD) is a disorder caused by the mutation in genes encoding for the cytochrome c oxidase enzyme (COX),1 a mitochondrial multimeric enzyme which acts as the terminal electron acceptor in the electron transport chain.2 COXD causes serious health issues in affected individuals, including severe muscle weakness, brain damage and disorders in organs such as heart, liver and kidneys.1 Diagnosis is mostly performed by muscle biopsy,3,4 which is invasive and time-consuming. Finding new effective and early diagnostic strategies is therefore crucial to minimize symptoms and long-term disabilities. This research proposes the use of scanning electrochemical microscopy (SECM) as a promising method to quantify COX activity in normal and COXD fibroblasts as cell models. We explore the interaction of the redox probe N,N,N’,N’-tetramethyl-p-phenylene diamine (TMPD) with COX as an indicator of enzymatic activity.5,6 A platinum ultramicroelectrode was used to monitor electrochemical currents at different scan speeds, which were later used to determine an apparent heterogeneous rate constant (k 0) for TMPD regeneration by each cell line using numerical modelling. A significant difference in k 0 values (p < 0.1) was observed when comparing normal and COXD fibroblasts. The further treatment of normal fibroblasts with a COX inhibitor, NaN3,7 induced significant decreases in k 0 (p < 0.01). The COX activity results determined using SECM were in good agreement with a standard spectrophotometric assay. Taken together, our findings confirm SECM as an effective and rapid method to detect COXD in living tissues and provides a foundation for the development of new assays to diagnose COXD in infants. References M. Brischigliaro and M. Zeviani, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1862, 148335 (2021).A. Timón-Gómez et al., Semin Cell Dev Biol, 76, 163–178 (2018).M. L. Simard, A. Mourier, L. C. Greaves, R. W. Taylor, and J. B. Stewart, Journal of Pathology, 245, 311–323 (2018).S. B. Wortmann, J. A. Mayr, J. M. Nuoffer, H. Prokisch, and W. Sperl, Neuropediatrics, 48, 309–314 (2017).S. Kuss, E. E. L. Tanner, M. Ordovas-Montanes, and R. G. Compton, Chem Sci, 8, 7682–7688 (2017).S. Thind et al., Proc Natl Acad Sci U S A (2023). In press.M. J. Fei et al., Acta Crystallogr D Biol Crystallogr, 56, 529–535 (2000).