THERMODYNAMIC ANALYSIS OF FLAVIN IN MITOCHONDRIAL NADH-UBIQUINONE OXIDOREDUCTASE (COMPLEX-I)

This paper reports the first direct characterization of flavin (noncov alently bound FMN) in energy coupling site I of the mitochondrial resp iratory chain. Thermodynamic parameters of its redox reactions were de termined potentiometrically monitoring the g = 2.005 signal of its fre e radical form in isolated bovine heart NADH:ubiquinone oxidoreductase (complex I). The midpoint redox potentials of consecutive one-electro n reduction steps are E(m)(1/0) = -414 mV and E(m)(2/1) = -336 mV at p H 7.5. This corresponds to a stability constant of the intermediate fl avosemiquinone state of 4.5 x 10(-2). The pK values of the free radica l (Fl <-> FlH(.)) and reduced flavin (FlH(-) <-> FlH(2)) were estimate d as 7.7 and 7.1, respectively. The potentiometrically obtained g 2.00 5 flavin free radical EPR signal revealed an unusually broad (2.4 mT) and pH-independent peak-to-peak line width. The spin relaxation of fla vosemiquinone in complex I is much faster than that of flavodoxin due to strong dipole-dipole interaction with iron-sulfur cluster N3. Guani dine, an activator of NADH-ferricyanide reductase activity of complex I, was found to have a strong stabilizing effect on the flavin free ra dical generated both by equilibration with the NADH/NAD(+) redox coupl e and by potentiometric redox titration. The addition of guanidine als o leads to a slight modification of the EPR spectrum of iron-sulfur cl uster N3. Anaerobic titration of flavosemiquinone free radical with th e strictly n = 2 NADH/NAD(+) and APADH/APAD(+) redox couples revealed that nucleotide binding narrows the EPR signal line width of the flavi n free radical to 1.7 mT and changes a shape of the titration curve. T his suggests a conformational change of the complex due to the substra te binding.