THE IRON-SULFUR CLUSTERS-2 AND UBISEMIQUINONE RADICALS OF NADH-UBIQUINONE OXIDOREDUCTASE ARE INVOLVED IN ENERGY COUPLING IN SUBMITOCHONDRIAL PARTICLES

The behavior of ubisemiquinone radicals and the iron-sulfur clusters 2 of NADH:ubiquinone oxidoreductase (Complex I) in coupled and uncouple d submitochondrial particles (SMP), oxidizing either NADH or succinate under steady-state conditions, was studied. Multifrequency EPR spectr a revealed that the two new g(z) lines of the clusters 2, only observe d during coupled electron transfer under conditions where energy dissi pation is rate-limiting [De Jong, A. M. Ph., Kotlyar, A. B., & Albrach t, S. P. J. (1994) Biochim. Biophys. Acta 1186, 163-171], are the resu lt of a spin-spin interaction of 2.8 mT. Investigation of the radical signals present in coupled SMP indicated that more than 90% of the rad icals can be ascribed to two types of semiquinones which are bound to Complex I (Q(I)-radicals) or ubiquinol:cytochrome c oxidoreductase (Co mplex III; Q(III)-radicals). The presence of Q(III)-radicals, but not that of Q(I)-radicals, was completely abolished by uncoupler. Part of the Q(I)-radicals weakly interact with the clusters 2 of Complex I. Th is uncoupler-sensitive interaction can amount to a splitting of the ra dical EPR signal of at most 1 mT, considerably weaker than the 2.8 mT splitting of the g(z) lines of the clusters 2. We propose that the 2.8 mT splitting of these g(z) lines results from an energy-induced spin- spin interaction between the two clusters 2 within the TYKY subunit of Complex I. The two clusters 2 show no interaction during electron tra nsfer in uncoupled SMP or in fully-reduced anaerobic-coupled SMP. The results point to a direct role of the Fe-S clusters 2 and the Q(I)-rad icals in the mechanism of coupled electron transfer catalyzed by Compl ex I.