Mitochondrial NADH fluorescence is enhanced by complex I binding

Abstract

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.

Figure 1

NADH oxidation and fluorescence enhancement occur in Clear Native PAGE bands associated with Complex I. 200 μg of mitochondrial preparation was loaded in each lane of the 3–12% native gel. A) Coomassie stained gel; B, C, D, and E are UV fluorescence images: B) after 5 min incubation in 100 μM NADH solution in the presence of oxygen; C) same as B, but 50μM DPI was added into the incubation media; D) after 4 h incubation in 100 μM NADH solution in an anaerobic environment; E) same as D, but 50μM DPI was added into the incubation media. F and G are NBT-stained gels, with (F) and without (G) the addition of DPI in to the media. Mass spectrometric protein identifications for CN-PAGE bands C1-C5 are provided in Table 1.

Figure 2

NADH/FMN fluorescence image consistent with Complex I oxidation of NADH. 200 μg of mitochondrial preparation was loaded in each lane of the 4–16% native gel. 100 mM NADH incubation for 10 minutes. A) Coomassie stained gel. B) 350 nm excitation (NADH fluorescence). C) 445 nm excitation (FMN fluorescence).

Figure 3

NADH fluorescence enhancement in Ghost Native PAGE. 100 μg of each purified Complex I (lane 1) and mitochondrial preparation (lanes 2, 3) were loaded onto the 3–12% native gel. A) Coomassie stained gel. B) UV fluorescence image of the GN-PAGE gel incubated overnight in 100 μM NADH solution in anaerobic chamber; C) same as B, but 50μM DPI was added into incubation media; D and E are NBT-stained gels, with (E) and without (D) addition of DPI into the media. Mass spectrum protein identifications for GN-PAGE bands G1–G4 can be found in Table 1.

Figure 4

No NADH fluorescence enhancement in denatured isolated subunits of Complex I. 40 μg of purified Complex I per well of SDS-gel was loaded. A) Coomassie stained gel. B) UV image of the SDS-gel incubated overnight in 100 μM NADH solution in an anaerobic chamber.

Figure 5

NADH fluorescence enhanced more efficiently by Complex I than by MDH binding First two lanes of 3–12% native gel were loaded with 47 μg (~50 pmol) of purified Complex I and the last three lanes were loaded with 8, 8, and 12 μg of MDH (~ 115, 115, and 170 pmol). Gel was incubated in 100 μM NADH overnight. A) Coomassie stained gel. B) UV fluorescence image.