Triplet state of the semiquinone-Rieske cluster as an intermediate of electronic bifurcation catalyzed by cytochrome bc1.

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 bc1 (Qo), for which detection of any intermediate states is extremely difficult. Here we discover a semiquinone at the Qo site (SQo) 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 (|J0| = 3500 MHz) indicates that at a lower magnetic field (typical of X-band) the SQo-FeS coupled centers can be described as a triplet state. Concomitantly with the appearance of the SQo-FeS triplet state, we detected a g = 2.0045 radical signal that corresponded to the population of unusually fast-relaxing SQo 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 bc1 monomers under aerobic conditions, challenging the paradigm of the high reactivity of SQo toward molecular oxygen. Recognition of stable SQo reflected in g = 1.94 and g = 2.0045 signals offers a new perspective on understanding the mechanism of Qo site catalysis. The frequency-dependent EPR transitions of the SQo-FeS coupled system establish a new spectroscopic approach for the detection of SQo in mitochondria and other bioenergetic systems.