NO-induced change in the oxidation state of cytochrome c in endothelial cells monitored by resonance Raman imaging

Nitric oxide (NO) is a key signalling molecule in endothelial cells, playing a complex role in the regulation of cell apoptosis. One of its critical molecular targets is cytochrome c (CytC), a heme protein whose oxidation state determines its function in mitochondrial respiration and cell death pathways. Using resonance Raman (RR) imaging with 405 nm excitation, we investigated NO-induced changes in the redox state of CytC in various endothelial cell lines. Calcium ionophore (A23187), known to activate endothelial NO synthase (eNOS), induced a shift in CytC from the ferrous (CytC-Fe^II) to ferric (CytC-Fe^III) state. This effect was cell type-dependent and most prominent in human aortic and dermal microvascular endothelial cells (HAECs, HMECs). The transition was prevented by L-NAME (NOS inhibitor), PEG-SOD (superoxide scavenger), and NecroX-5 (peroxynitrite scavenger), suggesting that ONOO^- plays a mediating role. Physiological NO inducers like VEGF and bradykinin also promoted CytC oxidation, but in a spatially distinct manner, suggesting subcellular specificity in NO signalling. Interestingly, applying an external NO donor (DEA-NONOate) did not trigger oxidation but briefly formed a CytC-Fe^II-NO complex. Despite mitochondrial membrane potential disturbances following A23187 treatment, no apoptosis was observed, indicating that CytC oxidation can be an early, reversible marker of mitochondrial stress rather than cell death. Our findings demonstrate the utility of RR imaging for real-time monitoring of CytC redox state and underscore the complexity of NO signalling in endothelial physiology and its potential implications for vascular health.