Mutation at the entrance of the quinone cavity severely disrupts quinone binding in respiratory complex I

Abstract

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.

Figure 1

Visualization of the Q-chamber and narrow bottleneck in complex I, with ubiquinone bound to complex I. Quinone is shown in blue, and the donut-shaped bottleneck is shown with surface rendering using PyMOL. The Q-chamber was calculated using the CAVER add-on.

Figure 2

Narrow bottleneck of wild type T. Thermophilus (PDB: 4HEA) (left), and of the AlaThr MUT (right). Alanine 63 (Alanine 52 in human complex I) is substituted with threonine (see ref , Ala52Thr in ND1). T. Thermophilus is our model system for all calculations, as it is similar to the human complex I bottleneck structure (see SI Figs. S3,S4). Importantly, the human bottleneck is not obstructed by any accessory subunits in the enzyme, so we anticipate the binding mechanism of Q to complex I to be comparable in both species. It is visually apparent that the substitution from alanine to threonine increases obstruction of the bottleneck.

Figure 3

The cross-section area distributions of the T. Thermophilus the wild type (a, left) and AlaThr (b, right) variant bottlenecks from two MD simulations. The first simulation restrains all bottleneck atoms near their positions in the crystal structure (crystal structure/rigid, red). The second simulation has zero position restraints on the bottleneck atoms (unrestrained/relaxed, green).

Figure 4

Bottleneck and quinone head group deformation energy during a series of steered MD simulations. The left column is the wild type bottleneck (green), and the right column is the AlaThr MUT bottleneck (red). The top row contains simulations performed with a rigid bottleneck, and bottom row contains simulations performed with a relaxed bottleneck. In each quadrant, the top graph is the cross-section area of the bottleneck, and the bottom graph is the bonded energy of the Q1 head group. Regions highlighted in blue roughly correspond to the time in the simulation when quinone is passing through the bottleneck. For comparability, note that the cross-section area in the top row span about 1 Ų, whereas the cross-section area in the bottom row span about 5 Ų.

Figure 5

Free energy of quinone passage through the bottleneck for the wild type WT (green) and AlaThr MUT variant (red) with uncertainties.

Figure 6

Free energy of water passage through the bottleneck when quinone is bound for the wild type (green) and AlaThr variant (red) with uncertainties. The free energy plotted here is from Eq. 1. We see there is a 15 kJ mol⁻¹ difference in the dissociation barrier between the two variants.

Figure 7

Demonstration of “clustering” in a representative steered MD simulation. A side profile of the bottleneck is rendered above, with the alanine 63 located at the top. Each point represents the position of the head group center of mass when the head group is slowly pulled through the bottleneck.

Figure 8

Graph of $\mathrm{\Delta }\mathrm{\Delta }G\left(\xi \right)$ (a, top), and DOS for both the WT and MUT variants (b, bottom). ξ is the same spline used for free energy calculation in Fig. 4. The DOS are approximated by projecting the position of the head group during a steered MD simulation onto ξ. There exist two barriers; the first peak is due to the head group’s entry to the bottleneck, and the second peak is due to the head group’s exit.