In all resolved structures of complex I, there exists a tunnel-like Q-chamber for ubiquinone binding and reduction. The entrance to the Q-chamber in ND1 subunit forms a narrow bottleneck, which is rather tight and requires thermal conformational changes for ubiquinone to get in and out of the binding chamber. The substitution of alanine with threonine at the bottleneck (AlaThr MUT), associated with 3460/ND1 mtDNA mutation in human complex I, is implicated in Leber's Hereditary Optic Neuropathy (LHON). Here, we show the AlaThr MUT further narrows the Q-chamber entrance cross-section area by almost 30%, increasing the activation free energy barrier of quinone passage by approximately 5 kJ mol^-1. This severely disrupts quinone binding and reduction as quinone passage through the bottleneck is slowed down almost tenfold. Our estimate of the increase in free energy barrier is entirely due to the bottleneck narrowing, leading to a reduction of the transition state entropy between WT and MUT, and thus more difficult quinone passage. Additionally, we investigate details of possible water exchange between the Q-chamber and membrane. We find water exchange is dynamic in WT but may be severely slowed in MUT. We propose that LHON symptoms caused by 3460/ND1 mtDNA mutation are due to slowed quinone binding. This leads to an increased production of reactive oxidative species due to upstream electron backup at the FMN site of complex I, thus resulting in a mt bioenergetic defect.

At the joint between the membrane and hydrophilic arms of the enzyme, the structure of the respiratory complex I reveals a tunnel-like Q-chamber for ubiquinone binding and reduction. The narrow entrance of the quinone chamber located in ND1 subunit forms a bottleneck (eye of a needle) which in all resolved structures was shown to be too small for a bulky quinone to pass through, and it was suggested that a conformational change is required to open the channel. The closed bottleneck appears to be a well-established feature of all structures reported so-far, both for the so-called open and closed states of the enzyme, with no indication of a stable open state of the bottleneck. We propose a squeeze-in mechanism of the bottleneck passage, where dynamic thermal conformational fluctuations allow quinone to get in and out. Here, using molecular dynamics simulations of the bacterial enzyme, we have identified collective conformational changes that open the quinone chamber bottleneck. The model predicts a significant reduction-due to a need for a rare opening of the bottleneck-of the effective bi-molecular rate constant, in line with the available kinetic data. We discuss possible reasons for such a tight control of the quinone passage into the binding chamber and mechanistic consequences for the quinone two-electron reduction.