Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I

Site-specific suppressors of superoxide production (named S1QELs) in the quinone-reaction site in mitochondrial respiratory complex I during reverse electron transfer have been previously reported; however, their mechanism of action remains elusive. Using bovine heart submitochondrial particles, we herein investigated the effects of S1QELs on complex I functions. We found that the inhibitory effects of S1QELs on complex I are distinctly different from those of other known quinone-site inhibitors. For example, the inhibitory potencies of S1QELs significantly varied depending on the direction of electron transfer (forward or reverse). S1QELs marginally suppressed the specific chemical modification of Asp(160) in the 49-kDa subunit, located deep in the quinone-binding pocket, by the tosyl chemistry reagent AL1. S1QELs also failed to suppress the binding of a photoreactive quinazoline-type inhibitor ([I-125]AzQ) to the 49-kDa subunit. Moreover, a photoaffinity labeling experiment with photoreactive S1QEL derivatives indicated that they bind to a segment in the ND1 subunit that is not considered to make up the binding pocket for quinone or inhibitors. These results indicate that unlike known quinone-site inhibitors, S1QELs do not occupy the quinone- or inhibitor-binding pocket; rather, they may indirectly modulate the quinone-redox reactions by inducing structural changes of the pocket through binding to ND1. We conclude that this indirect effect may be a prerequisite for S1QELs' direction-dependent modulation of electron transfer. This, in turn, may be responsible for the suppression of superoxide production during reverse electron transfer without significantly interfering with forward electron transfer.