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)