Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

Abstract

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.

Pulse frequency dependency of photobiomodulation on the bioenergetic functions of human dental pulp stem cells

“…Considering previous reports 27 , 34 – 39 that showed a relationship between mitochondrial activity and DL, we used rotenone, which inhibits the transfer of electrons from iron-sulfur centers in mitochondrial complex I to ubiquinone, or antimycin-A (AMA), which inhibits the oxidation of ubiquinone in mitochondrial complex III. In both cases, time delays after CW-PBM treatment became slower, but those after PW-PBM treatments were faster than in cells untreated by chemicals (Fig.…”

Synergic pro-apoptotic effects of Ferulic Acid and nanostructured lipid carrier in glioblastoma cells assessed through molecular and Delayed Luminescence studies

“…So, the multimodal behaviour observed in DL time trends in the red region can be due to more classes of light-emitting states. The decrease of DL yield at shorter time (DL-I 650 /DLI 650 ) and the increase at longer times (DL-II 650 /DLI 650 and DL-III 650 /DLI 650 ) could be ascribed to, respectively: i) the oxygen-dependent quenching of DL emitted from PpIX 24,25,70 ; ii) the enhancement of the dimol photoemission generated by colliding molecules of singlet oxygen produced at Complex I of MRC 26 . On the contrary, DL-II 650 /DLI 650 and DL-III 650 /DLI 650 decrease in FA-NLCs U-87 MG treated cells could be explained looking to the scavenger activity towards free radicals of FA when it was carried into mitochondrion through NLCs.…”

“…Till now the studies regarding DL from mammalian cells highlighted that mitochondria could represent the primary source of this emission [21][22][23][24] . In particular, a connection between DL characteristics and functionality of the MRC-Complex I has been suggested 26 . For this reason, DL spectral emissions have been evaluated in the blue, green/yellow and red regions where mitochondrial biomarkers, such as reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide (FMN), protoporphyrin IX and singlet oxygen ( 1 O 2 ) dimols emit [60][61][62] .…”

“…To better analyse and compare different DL kinetics, it resulted useful, as reported in previous papers 22,23 , consider, inside each time decay, the DL-integrals corresponding to the time intervals 10-100 µs, 100 µs-1 ms and 1-10 ms, denoting them DL-I, DL-II and DL-III respectively. This analysis reflects the presence of different emitting species, characterized by different lifetimes, that could be related to different electron transfer steps in MRC Complex I 26 . More precisely each DL-integrals, related to a certain time interval of the decay, was normalized to the corresponding DLI value related to the whole time interval (10 µs −10 ms) of the decay, that is, in the case of emission in blue region, the ratios DL-I 450 /DLI 450, DL-II 450 /DLI 450, DL-III 450 /DLI 450 were analysed.…”

Effects of Ion Irradiation on Seedlings Growth Monitored by Ultraweak Delayed Luminescence

“…The delayed luminescence of seeds was measured by using an improved version of ARETUSA set-up developed at LNS-INFN and able to study the spectral properties of the DL issued by individual seeds [21]. …”