Thermodynamic analysis of flavin in mitochondrial NADH:ubiquinone oxidoreductase (complex I).
This paper reports the first direct characterization of flavin (noncovalently bound FMN) in energy coupling site I of the mitochondrial respiratory chain. Thermodynamic parameters of its redox reactions were determined potentiometrically monitoring the g = 2.005 signal of its free radical form in isolated bovine heart NADH:ubiquinone oxidoreductase (complex I). The midpoint redox potentials of consecutive one-electron reduction steps are Em1/0 = -414 mV and Em2/1 = -336 mV at pH 7.5. This corresponds to a stability constant of the intermediate flavosemiquinone state of 4.5 x 10(-2). The pK values of the free radical (Fl.-<==>FlH.) and reduced flavin (FlH-<==>FlH2) were estimated as 7.7 and 7.1, respectively. The potentiometrically obtained g = 2.005 flavin free radical EPR signal revealed an unusually broad (2.4 mT) and pH-independent peak-to-peak line width. The spin relaxation of flavosemiquinone in complex I is much faster than that of flavodoxin due to strong dipole-dipole interaction with iron-sulfur cluster N3. Guanidine, an activator of NADH-ferricyanide reductase activity of complex I, was found to have a strong stabilizing effect on the flavin free radical generated both by equilibration with the NADH/NAD+ redox couple and by potentiometric redox titration. The addition of guanidine also leads to a slight modification of the EPR spectrum of iron-sulfur cluster N3. Anaerobic titration of flavosemiquinone free radical with the strictly n = 2 NADH/NAD+ and APADH/APAD+ redox couples revealed that nucleotide binding narrows the EPR signal line width of the flavin free radical to 1.7 mT and changes a shape of the titration curve.(ABSTRACT TRUNCATED AT 250 WORDS)
Sled
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Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Rudnitzky
N I
NI
Hatefi
Y
Y
Ohnishi
T
T
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005609:Free Radicals; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry*; D010084:Oxidation-Reduction; D011199:Potentiometry; D013816:Thermodynamics
D005609:Free Radicals; D006146:Guanidines; D007506:Iron-Sulfur Proteins; D009243:NAD; D005486:Flavin Mononucleotide; D016660:NAD(P)H Dehydrogenase (Quinone); D019791:Guanidine