Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

• Published in: Journal of biomedical optics Vol. 18; no. 12; p. 127006
• Main Authors: Baran, Irina, Ionescu, Diana, Privitera, Simona, Scordino, Agata, Mocanu, Maria Magdalena, Musumeci, Francesco, Grasso, Rosaria, Gulino, Marisa, Iftime, Adrian, Tofolean, Ioana Teodora, Garaiman, Alexandru, Goicea, Alexandru, Irimia, Ruxandra, Dimancea, Alexandru, Ganea, Constanta
• Format: Journal Article
• Language: English
• Published: United States Society of Photo-Optical Instrumentation Engineers 01.12.2013
• Subjects: Apoptosis - drug effects; Cell Survival - drug effects; Electron Transport Complex I - chemistry; Electron Transport Complex I - drug effects; Electron Transport Complex I - metabolism; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Humans; Hydrogen Peroxide - pharmacology; Inhibitors; Jurkat Cells; Kinetics; Luminescence; NAD - chemistry; NAD - metabolism; NADH; NADP - chemistry; NADP - metabolism; Nicotinamide adenine dinucleotide; Phosphates; Pyridine nucleotides; Reduction; Rotenone - pharmacology; Spectrometry, Fluorescence - methods; Spectroscopy; Uncoupling Agents - pharmacology; flavin mononucleotide; electron transfer; reduced nicotinamide adenine dinucleotide; menadione; delayed luminescence; rotenone; complex I; quercetin
• ISSN: 1083-3668; 1560-2281
• DOI: 10.1117/1.JBO.18.12.127006

The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.