Triplet State of the Semiquinone–Rieske Cluster as an Intermediate of Electronic Bifurcation Catalyzed by Cytochrome bc(1)

[Image: see text] Efficient energy conversion often requires stabilization of one-electron intermediates within catalytic sites of redox enzymes. While quinol oxidoreductases are known to stabilize semiquinones, one of the famous exceptions includes the quinol oxidation site of cytochrome bc(1) (Q(o)), for which detection of any intermediate states is extremely difficult. Here we discover a semiquinone at the Q(o) site (SQ(o)) that is coupled to the reduced Rieske cluster (FeS) via spin–spin exchange interaction. This interaction creates a new electron paramagnetic resonance (EPR) transitions with the most prominent g = 1.94 signal shifting to 1.96 with an increase in the EPR frequency from X- to Q-band. The estimated value of isotropic spin–spin exchange interaction (|J(0)| = 3500 MHz) indicates that at a lower magnetic field (typical of X-band) the SQ(o)–FeS coupled centers can be described as a triplet state. Concomitantly with the appearance of the SQ(o)–FeS triplet state, we detected a g = 2.0045 radical signal that corresponded to the population of unusually fast-relaxing SQ(o) for which spin–spin exchange does not exist or is too small to be resolved. The g = 1.94 and g = 2.0045 signals reached up to 20% of cytochrome bc(1) monomers under aerobic conditions, challenging the paradigm of the high reactivity of SQ(o) toward molecular oxygen. Recognition of stable SQ(o) reflected in g = 1.94 and g = 2.0045 signals offers a new perspective on understanding the mechanism of Q(o) site catalysis. The frequency-dependent EPR transitions of the SQ(o)–FeS coupled system establish a new spectroscopic approach for the detection of SQ(o) in mitochondria and other bioenergetic systems.