Research Article

The coupling mechanism of mammalian respiratory complex I

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Science  30 Oct 2020:
Vol. 370, Issue 6516, eabc4209
DOI: 10.1126/science.abc4209

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Secrets of a proton pumping machine

Mitochondrial complex I serves as a primary entry point for electrons from the tricarboxylic acid cycle into the mitochondrial electron transport chain. This massive, membrane-embedded protein complex must couple quinone reduction to conformational changes across more than 150 angstroms within four separate proton pumps. Kampjut et al. determined five structures of complex I in states along the catalytic cycle, a deactive conformation, and one with the inhibitor rotenone bound. The resolution of some structures was sufficient to see water molecules and to trace putative paths for proton transfer within the proton-pumping membrane domain. The structures add valuable details that provide a basis for generating mechanistic hypotheses for this crucial complex.

Science, this issue p. eabc4209

Structured Abstract

INTRODUCTION

Complex I is the first and, with 45 subunits and a total mass of ~1 MDa, the most elaborate of the mitochondrial electron transfer chain enzymes. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that drives the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell. In each catalytic cycle, the transfer of two electrons from nicotinamide adenine dinucleotide (NADH) to a hydrophobic electron carrier quinone, which happens in the peripheral arm of the enzyme, is coupled to the translocation of four protons across the inner mitochondrial membrane in the membrane arm. The exact mechanism of this energy conversion currently presents an enigma because of complex I’s size and the spatial separation between the two reactions.

RATIONALE

To understand the coupling mechanism of complex I, we solved its cryo–electron microscopy (cryo-EM) structures in five different conditions, including the substrate- and inhibitor-bound states and during active turnover, unlocking the various conformations that the enzyme goes through during the catalytic cycle. We also improved the resolution to up to 2.3 to 2.5 Å, allowing us to directly observe water molecules critical for proton pumping.

RESULTS

We showed that opening and closing movements of the peripheral and membrane arms of complex I are critical for catalysis. Opening and closing is accompanied by coordinated conformational changes at the junction between the two arms, around the quinone binding cavity. These changes involve five conserved protein loops and are initiated by the reduction of quinone, the resulting negative charge in its cavity, and decylubiquinone (DQ) movement between the deep and the shallow binding sites. The bulky inhibitor rotenone also binds at these two sites and, unexpectedly, also within ND4—one of the three antiporter-like subunits. The deactive state is defined by a notable relocation of the entire ND6 transmembrane (TM) helix 4, arresting the enzyme in the open conformation.

The PSST and 49-kDa subunit loops need to be ordered in the retracted state to enable quinone reduction as observed in the turnover closed class. Upon enzyme reduction with NADH in the open state, the 49-kDa loop extends into the cavity and the ND1 subunit loop flips upward, thus ejecting the reduced quinol. Conformational changes of the ND1 and ND3 loops also transmit the conformational changes in the quinone cavity to the rest of the enzyme (labeled “coupling” in the figure) by influencing the open-closed transition in the E-channel (the proton channel nearest to the quinone site). Entire TM helices of the ND1 subunit tilt upon opening, leading to a notable rotation of the ND6 TM3 helix, accompanied by the formation of the π-bulge.

Crucially, the rotation of this helix controls the formation of a critical water wire, which delivers protons from the conserved glutamates in subunit ND4L to the quinone site. This key feature brings the “charge action” of the quinone reaction directly next to ND2, the first out of the three homologous antiporter-like subunits, initiating a “wave” of electrostatic interactions propagating to the distal antiporter ND5. Analysis of water networks and charge distribution in the closed and open states of complex I under turnover explains how the protons are translocated in these waves within the antiporters and how this is coordinated between the four separate proton pumps and quinone reduction. A key role in this process is played by electrostatic interactions between the conserved charged residues, forming the highly hydrated “central axis” of the membrane arm. The distribution of the observed water molecules also suggests that links to the matrix and intermembrane space (IMS) sides in the distal subunit ND5 are much more hydrated than in other antiporters, and we propose the possibility that all four protons per cycle are ejected into the IMS via this subunit, rather than one per each antiporter (dashed arrows in the figure).

CONCLUSION

A comparison of conformational changes induced by substrate binding, turnover, inhibition, and deactivation allowed us to propose a detailed mechanistic model of the entire catalytic cycle in mammalian complex I, which combines elements of conformational (quinone site–E-channel) and electrostatic (antiporters) coupling.

A roadmap to the binding sites, proton pathways, and coupling mechanism of complex I.

A cross section of the cryo-EM density of complex I during turnover reveals an intricate machinery involved in catalysis. The solid black arrows show the propagation of the conformational changes and electrostatic interactions during the catalytic cycle; the gray arrows show the proton translocation pathways, with dashed arrows indicating the less likely paths. Core subunits are colored, and supernumerary subunits are in gray.

Abstract

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo–electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

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