ubiquinone and iodoacetamidoerythrosin

The coenzyme quinone (CoQ) binding region of mitochondrial NADH:CoQ reductase (complex-I) was investigated by the fluorescent probes erythrosine-5'-iodoacetamide (ER) and 3,3'-diethyloxadicarbocyanine iodide (DODCI). Both steady-state and time-resolved fluorescence was used in these experiments. Both probes competed for the binding site of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), an analogue of CoQ. The fluorescence lifetimes of the complex-I bound probes were approximately 600 ps and approximately 1.7 ns in the cases of ER and DODCI, respectively. Binding of the probes was not affected by the binding of the inhibitor rotenone. However, rotenone binding caused some changes in the lifetime of the bound probes. Reduction of the enzyme caused an increase in the level of binding of ER and a decrease in the level of binding of DODCI. The level of binding of cationic DODCI increased with the increase in pH, and in the case of anionic of ER the trend was reverse. Binding of Ca2+ to complex-I resulted in an increase in the level of binding of ER and a decrease in the level of binding of DODCI. Reaction with N,N'-dicyclohexylcarbodiimide (DCCD) resulted in alterations in the time-resolved fluorescence profiles of dye: complex-I system. All these results were interpreted as due to the presence of carboxyl group(s) with pKa approximately 6 in the probe/CoQ binding region. The rotational correlation time (tau r) of DODCI bound at the CoQ region was 2-3 ns.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acids; Amines; Animals; Binding Sites; Binding, Competitive; Carbocyanines; Cations, Divalent; Cattle; Electron Transport; Electron Transport Complex I; Erythrosine; Fluorescence Polarization; Fluorescent Dyes; Mitochondria, Heart; Models, Chemical; NADH, NADPH Oxidoreductases; Spectrometry, Fluorescence; Ubiquinone

The fluorescent probe erythrosine 5'-iodoacetamide (ER) binds to mitochondrial NADH-CoQ reductase (Complex-I) accompanied by an enhancement of the fluorescence intensity. The binding of the CoQ analogue, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), decreased the fluorescence intensity of the ER:Complex-I system. The 'site 1' inhibitor rotenone did not decrease the fluorescence intensity showing the non-identical nature of the binding sites of DB and rotenone. Also, the reduced form of DB did not decrease the fluorescence intensity. The decrease of the fluorescence intensity by DB was shown to be due to the removal of bound ER by DB. The rapid kinetics of ER binding was studied by temperature-jump relaxation. While DB caused complete elimination of the relaxation process in the ER:Complex-I system, rotenone caused only a decrease in the relaxation rate, suggesting conformational change. The relaxation rate showed a pH dependence with a maximum around pH 7.5.

Topics: Binding Sites; Biological Transport; Coenzymes; Erythrosine; Fluorescent Dyes; Kinetics; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Protons; Rotenone; Ubiquinone