Label‐free tracking of cytochrome C oxidation state in live cells by resonance Raman imaging

• Published in: FEBS letters 2024-08, Vol.598 (16), p.1981-1988
• Main Authors: Szczesny‐Malysiak, Ewa, Bartkowiak, Amanda, Dybas, Jakub
• Format: Article
• Language: English
• Subjects: Animals; cytochrome C; Cytochromes c - metabolism; heme iron oxidation state; Humans; Mitochondria - metabolism; Oxidation-Reduction; resonance Raman imaging; Spectrum Analysis, Raman - methods
• ISSN: 0014-5793; 1873-3468; 1873-3468
• DOI: 10.1002/1873-3468.14905

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt‐FeII) and oxidized ferric (CytC‐FeIII) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC‐FeIII to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt‐FeII to CytC‐FeIII within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear‐cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label‐free detection of ferrous and ferric heme proteins. A novel, label‐free methodology based on resonance Raman imaging with 405 nm excitation wavelength was uniquely designed to detect and distinguish between ferrous (FeII) and ferric (FeIII) heme proteins. The experiments were performed on live endothelial cells stimulated to produce NO which led to an increase in ferric cytochrome C.