Characterization of a semiquinone radical bound in the active site of respiratory complex I by hyperfine EPR spectroscopy

Quinone reduction is a key step in catalysis by respiratory complex I (NADH:quinone oxidoreductase, RCI), an essential energy-transducing enzyme that drives ATP synthesis by oxidative phosphorylation. However, the mechanism of quinone reduction and the role of semiquinone (SQ) intermediates in catalysis remain poorly understood. Spectroscopic investigations of SQ species have been complicated by difficulties in assigning the low-intensity g~2 EPR signals observed using native membrane systems or non-native quinone species. Here, we employ a biochemically and spectroscopically 'clean' proteoliposome system to investigate SQ formation by mammalian RCI in the absence of other respiratory proteins and using the native ubiquinone-10 (Q10) substrate. Following addition of NADH, we observe a g = 2.005 signal and use continuous-wave EPR to assign it to a SQ radical bound at the top of the quinone-binding channel, close to iron-sulfur cluster N2, the terminal cluster in the chain that connects the two substrate-binding sites. Using an EPR probehead modified for investigation of low-intensity signals, we then employ a hyperfine pulse EPR technique (HYSCORE) to define the coordination environment of the SQ species, including a direct hydrogen bond with a histidine Nδ-H, which we assign to the conserved active-site histidine essential for catalysis. Our results shed light on the Q10 reduction reaction that is key to the mechanism of energy coupling in RCI, and demonstrate how pulse EPR methods can provide detailed information on radical species formed in challenging energy-converting membrane proteins.