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.

Reduced metabolic rate and oxygen radicals production in stored insect sperm

“…Total NAD(P)H decreases with increasing ATP production, but free NAD(P)H is used up first [21]. Consequently, the proportion of bound-NAD(P)H relative to total NAD(P)H increases with increasing metabolic rate.…”

NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

“…Reflected light was band pass filtered at 460 Ϯ 20 nm to image endogenous NADH fluorescence and transmitted light was band pass filtered at 585 Ϯ 10 nm to image CICF fluorescence. Because NADH bound to Complex I of the ETC results in an amplification of the NADH fluorescence, captured fluorescence was assumed to be of mitochondrial origin (3). Addition of CICF did not increase the 460 Ϯ 20 nm filtered fluorescence signal, indicating no crosstalk of CICF into the NADH signal.…”

Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell

“…5). Although studies by Blinova et al 32,38 suggested that the NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence intensity, the association between the sum expression of 5 complex proteins, not Complex I, and τ 1 is significant (R 2 ¼ 0.92, p ¼ 0.01) as shown in Table 1. Complex III is marginally associated with τ 1 (R 2 ¼ 0.83, p ¼ 0.05).…”

“…NADH was known to strongly associate with respiration enzyme Complex I. 32 The free/bound NADH ratio was suggested to dominate the change of NADH fluorescence lifetime. 31,33 Therefore, our observed increase in fluorescence NADH lifetime during osteogenic differentiation of hMSCs was possibly attributed to NADH interacting with respiration enzymes complex proteins, particularly Complex I.…”