Resolving Chemical Dynamics in Biological Energy Conversion: Long-Range Proton-Coupled Electron Transfer in Respiratory Complex I

Abstract

Biological energy conversion is catalyzed by membrane-bound proteins that transduce chemical or light energy into energy forms that power endergonic processes in the cell. At a molecular level, these catalytic processes involve elementary electron-, proton-, charge-, and energy-transfer reactions that take place in the intricate molecular machineries of cell respiration and photosynthesis. Recent developments in structural biology, particularly cryo-electron microscopy (cryoEM), have resolved the molecular architecture of several energy transducing proteins, but detailed mechanistic principles of their charge transfer reactions still remain poorly understood and a major challenge for modern biochemical research. To this end, multiscale molecular simulations provide a powerful approach to probe mechanistic principles on a broad range of time scales (femtoseconds to milliseconds) and spatial resolutions (10¹-10⁶ atoms), although technical challenges also require balancing between the computational accuracy, cost, and approximations introduced within the model. Here we discuss how the combination of atomistic (aMD) and hybrid quantum/classical molecular dynamics (QM/MM MD) simulations with free energy (FE) sampling methods can be used to probe mechanistic principles of enzymes responsible for biological energy conversion. We present mechanistic explorations of long-range proton-coupled electron transfer (PCET) dynamics in the highly intricate respiratory chain enzyme Complex I, which functions as a redox-driven proton pump in bacterial and mitochondrial respiratory chains by catalyzing a 300 Å fully reversible PCET process. This process is initiated by a hydride (H^-) transfer between NADH and FMN, followed by long-range (>100 Å) electron transfer along a wire of 8 FeS centers leading to a quinone biding site. The reduction of the quinone to quinol initiates dissociation of the latter to a second membrane-bound binding site, and triggers proton pumping across the membrane domain of complex I, in subunits up to 200 Å away from the active site. Our simulations across different size and time scales suggest that transient charge transfer reactions lead to changes in the internal hydration state of key regions, local electric fields, and the conformation of conserved ion pairs, which in turn modulate the dynamics of functional steps along the reaction cycle. Similar functional principles, which operate on much shorter length scales, are also found in some unrelated proteins, suggesting that enzymes may employ conserved principles in the catalysis of biological energy transduction processes.

Figure 1

(a) Structure and function of Complex I. Reduced NADH donates electrons to the 100 Å long FeS chain that transfers them to quinone (Q). Q is reduced to quinol (QH₂), which triggers the transfer of four protons across the 200 Å long membrane domain. Point mutations of residues in the proton pumping subunits (shown in blue, red, yellow, gray/green) lead to inhibition of the Q oxidoreduction activity. (b) Multiscale simulation approaches can be used for probing the structure, function, and dynamics of PCET mechanisms in Complex I and other energy transducing enzymes. QM/MM models (left) allow exploring the local electronic structure, energetics, and dynamics on picosecond time scales (QM, QM region; MM, MM region; L, link atom in pink); atomistic MD (aMD) simulations (middle) enable sampling of the microsecond dynamics in a model of the biochemical environment; whereas coarse-grained models (cgMD) (right, showing a 1:4 mapping of beads:heavy atoms) allow exploring the micro- to millisecond time scale, but with loss of atomic detail. (c) The systems can be explored by unbiased MD simulations, potential energy surface (PES) scans, or free energy sampling methods at the different theory levels. The MD simulations allow probing, e.g., the dynamics of a reaction coordinate over time (here proton transfer, PT), whereas PES scan or FE sampling allows computing free energy/energy profiles along a reaction coordinate of interest (here a PT reaction).

Figure 2

Multiscale simulations of PCET reactions in Complex I. (a) PCET between NADH and FMN leads to hydride (H^–) transfer between the cofactors, followed by ET to the nearby FeS centers. (b) Energy profiles, spin, and charge analysis of the cofactors along the PCET process and coupled ET (here to N1a) along a proton transfer reaction coordinate. All FeS center were initially modeled in their oxidized state. The change in redox state of N1a is indicated in the top panel. (c) ET pathway down along the 100 Å FeS chain to Q. The figure also shows water molecules surrounding the FeS clusters. (d) QM/MM model of the Q oxidoreduction process: PCET between the terminal N2 FeS and Q triggers PT from nearby Tyr and His residues. (e) QM/MM MD of the PCET process shows the reversible formation of the QH₂ by ET from the reduced N2 FeS center, and vice versa. Reproduced from refs (4) and (41). Copyright 2017 and 2019, respectively, American Chemical Society.

Figure 3

(a) Quinol formation in the active site (site 1) leads to dissociation of the QH₂ to a second binding region (site 2). The process is coupled to conformational changes in charged residues and surrounding loop regions. (b) CryoEM map for piericidin A and modeled end-on binding mode of two inhibitors bound in the Q-cavity. Data from ref (55). (c) Conformational changes between transmembrane helices (e.g., TM3 of ND6) in the membrane domain of Complex I regulate the formation of (d) proton conducting water wires (marked in red circle). (e) Free energy profiles computed based on QM/MM US simulations (profile) and QM cluster models (energy levels based on reaction pathway optimization) for the PT from the E-channel region to residues at the ND2 interface. The amino acid binding the proton is marked in the free energy profile. R₁ and R₂ are reaction coordinates used to model the PT reactions (see ref (2) for further details). Reproduced with permission from ref (2). Copyright 2021 the Authors. Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/.

Figure 4

(a) Microsecond MD simulations predict S-shaped proton pathways across the membrane (averaged water structure). (b) Comparison of the water wires based on MD simulations (water in red spheres) and cryoEM experiments (in purple). Data from ref (61). Structure of the charged array along the center of the membrane domain is highlighted with Lys(Arg) (spheres in blue) and Glu(Asp) (in red), and His (in gray). (c) QM/MM free energy calculations of PT along a predicted water wire in the antiporter-like subunits with open and closed ion pairs. The PT reaction coordinate is defined as a linear combination of bond-forming and bond-breaking distances along the PT reaction from K235 to E377 (see ref (3) for further details). (d) Ion pair (IP) opening enables lateral PT within the antiporter-like subunits. Protonation of the terminal residue favors opening of the IP in the next subunit. Green signal, favorable PT; red signal, unfavorable PT. Adapted from refs (1) and (3). Copyright 2020 American Chemical Society.

Figure 5

Schematic representation of the putative long-range PCETmechanism in Complex I. (a) NADH-driven ET along the FeS chain reduces Q to QH₂, which triggers conformational changes and motion of the QH₂ to a second binding site. The QH₂/QH^– initiate stepwise PT reactions that lead to consecutive opening/conformational changes of ion pairs, and modulate the energetics of lateral PT reactions. The signal propagates to the terminal edge of ND5 (in red). (b) Proton release across the membrane increases the pK_a of the middle Lys, leading to H⁺ uptake from the bulk (H⁺ in red) and closure of the IP in the last subunit (subunit in red). The closed IP destabilizes the proton stored at the ND4/ND5 interface that is ejected across the membrane. The signal propagates “backward” in a similar way via ND4 (in blue), ND2 (in yellow), and ND4L/ND1(orange/green) to the quinol, which is ejected to the membrane. The new reaction cycle is initiated by reprotonation of the Q-active site and uptake of a new Q from the membrane. Adapted from ref (3). Copyright 2020 American Chemical Society.

Figure 6

Comparison of functional elements involved in CT/PCET reaction in different enzymes. (a) Complex I (closeup of antiporter-like subunit ND4). (b) Cytochrome c oxidase (nonpolar cavity around the active site). (c) Photosystem II (vicinity of the CaMn₄O₅ cluster). (d) ATPase reaction in Hsp90 (active site in the N-terminal domain) (see main text). (e) Designed buried ion pairs in artificial bundle proteins, showing different ion-pair conformations, with aims to understand functional principles from a bottom-up approach. All ion pairs shown are located in buried core regions of the proteins. Panel (a) adapted from ref (1). Copyright 2020 American Chemical Society. Panel (e) adapted with permission from ref (67). Copyright 2021 the Authors. Published by Springer Nature under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/.