Hypothesis the mecanism of ATP synthase conformational change by rotation of the b-subunit

In a majority of living organisms, F o F 1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP þ P i → ATP þ H 2 O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-O ADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar PO 3 − ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.quantum mechanics | reaction mechanism | molecular motor T he F o F 1 -ATP synthase is vital for cell energy production, being responsible for most of the ATP synthesis in membranes of bacteria, chloroplasts, and mitochondria. This multisubunit enzyme complex has high structural similarity among different species. It produces ATP as a biological nanomotor driven by an electrochemical potential difference across membranes as part of respiration or photosynthesis (1). The active sites are located in the F 1 domain, which in Escherichia coli consists of α 3 β 3 γδϵ subunits, three αβ-pairs surrounding a central α-helical coiled coil γ-subunit. The ATP synthesiswith P i ¼ H 2 PO 4 − , occurs at catalytic sites formed in the three β-subunits at the α/β-interfaces (2). The most prominent model assumes that the ATP-producing reaction occurs due to a rotation of the γ-shaft, inducing conformational changes in the surrounding αβ-subunit pairs according to the unbinding and rotational coupling mechanism (3-7). It was also demonstrated that excess ATP may be hydrolyzed by isolated F 1 resulting in reverse rotation of the γ-subunit (7). In the first crystal structure of F 1 -ATPase (3), the three catalytic sites contained a nonhydrolyzable ATP analogue in one site, an ADP in the other, the third site being empty. Because of this observation, also supported by ATP binding affinity measurements (8, 9), the sites were named "tight binding site" (β TP ), "loose binding site" (β DP ), and "empty site" (β E ). According to the trisite mechanism model, during ATP synthesis each of the three αβ-subunit pairs changes conformation along the repeating cycle, β E → β DP → β TP → β E...

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confidence: 99%

Double-lock ratchet mechanism revealing the role of αSER-344 in F _o F ₁ ATP synthase

Beke‐Somfai

Lincoln²,

Nordén³

2011

“…F 1 -ATPase, the catalytic part of the energy conversion enzyme F 0 F 1 -ATP synthase, is known to act as a rotary motor upon ATP hydrolysis (19)(20)(21)(22)(23)(24). Single-molecule experiments have visualized the rotation and elucidated some of the details; the ␥-subunit rotates unidirectionally in discrete 120°steps with each ATP hydrolysis (25), and the 120°step can be divided into 90°a nd 30°substeps at lower ATP concentration (26).…”

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confidence: 99%

Folding-based molecular simulations reveal mechanisms of the rotary motor F ₁ –ATPase

Koga

Takada

2006

120

146

Biomolecular machines fulfill their function through large conformational changes that typically occur on the millisecond time scale or longer. Conventional atomistic simulations can only reach microseconds at the moment. Here, extending the minimalist model developed for protein folding, we propose the ''switching Go model'' and use it to simulate the rotary motion of ATP-driven molecular motor F 1-ATPase. The simulation recovers the unidirectional 120°rotation of the ␥-subunit, the rotor. The rotation was induced solely by steric repulsion from the ␣3␤3 subunits, the stator, which undergoes conformation changes during ATP hydrolysis. In silico alanine mutagenesis further elucidated which residues play specific roles in the rotation. Finally, regarding the mechanochemical coupling scheme, we found that the tri-site model does not lead to successful rotation but that the alwaysbi-site model produces Ϸ30°and Ϸ90°substeps, perfectly in accord with experiments. In the always-bi-site model, the number of sites occupied by nucleotides is always two during the hydrolysis cycle. This study opens up an avenue of simulating functional dynamics of huge biomolecules that occur on the millisecond time scales involving large-amplitude conformational change.always-bi-site model ͉ energy landscape ͉ funnel ͉ switching Go model B iomolecular machines, such as the ribosome, transporter, and molecular motors, fulfill their function through largeamplitude conformational change. Structural information before and after the conformational change has been provided by x-ray crystallography and other methods for many cases (1). However, these methods do not directly observe the molecular dynamics that connects two-end structures. These dynamical aspects can be observed directly by fluorescence and other time-resolved spectroscopy; however, the latter methods monitor local structure but do not give global structural information. In this sense, molecular dynamics simulation is potentially powerful because it can provide full time-dependent structural information about biomolecular machines (2-5). Yet, functional cycles of these systems typically take milliseconds or longer, which is far beyond the current reach of molecular simulations with all-atom standard force-fields (6). Thus, a complementary approach may be to coarse-grain the molecular representation, thereby enabling the simulation orders of magnitude-longer time scales.Coarse-graining drops some details from the model, while hopefully keeping the essence. To achieve them, we need some perspective on the physical process we are trying to simply. Upon conformational change, some portions of molecules do not significantly change their structures; here, the atomic motion can be well approximated by harmonic fluctuations. It has been known that very simple C ␣ models, such as the elastic network model (7), can reproduce low-frequency harmonic fluctuation fairly well. Conversely, large-scale conformational change involves rearrangement of nonlocal contacts between amino acids, which is...

“…Hence any multiplesite mechanism requires a shift in the coupling of Fo to each of the catalytic sites in turn. Such a sequential linking of the proton channel to active sites has been suggested to involve rotation of parts of F1 with respect to Fo (11,12,19), producing a changing structural as well as functional asymmetry in the complex. However, evidence for motion during coupling has been lacking.…”

Section: Introductionmentioning

confidence: 99%

Ligand-dependent structural variations in Escherichia coli F1 ATPase revealed by cryoelectron microscopy.

Gogol

Johnston

Aggeler

et al. 1990

100

The Escherichia coli F, ATPase, ECFI, has been examined by cryoelectron microscopy after reaction with Fab' fragments generated from monoclonal antibodies to the a and E subunits. The enzyme-antibody complexes appeared triangular due to the superposition of three anti-a Fab' fragments on alternating densities of the hexagonally arranged a and .3 subunits. The Fab' to the E subunit superimposed on a .8 subunit. A density was observed near the center of the structure in the internal cavity. Mg2+ were added and ATP hydrolysis was allowed to proceed, almost two-thirds of the images were in the class in which the central density was closest to the 13 subunit superimposed by the E subunit. We conclude that domains within the ECF, structure, either the central mass or a domain including the E subunit, move in the enzyme in response to ligand binding. We suggest that this movement is involved in coupling catalytic sites to the proton channel in the FO part of the ATP synthase.Energy-transducing membranes, including the plasma membrane of bacteria, the thylakoid membrane of chloroplasts, and the mitochondrial inner membrane, all contain a structurally homologous F1Fo ATP synthase (reviewed in refs. 1 and 2). This enzyme complex uses the energy of the transmembrane proton gradient, generated by oxidative or photophosphorylation, to synthesize ATP from ADP and inorganic phosphate (Pi) (3,4).The ATP synthase is organized into two distinct subassemblies, a catalytically active portion extrinsic to the membrane (F1) and a bilayer-spanning proton channel (FO). The compositionally simplest ATP synthases are found in bacteria: the Escherichia coli F1 (ECF1) is made up of five subunits, a, /3, y, 8, and E, in the ratio 3:3:1:1:1, and the ECFo is composed of three subunits, a, b, and c, in a stoichiometry of 1:2:10-12 (4-7).The catalytic sites in F1 are located on the ,8 subunits (1, 2); hence there are potentially three active sites per F1. Catalysis is highly cooperative, in that release of products is very slow when only one site is occupied, and increases dramatically (up to 106-fold) by binding substrate at a second catalytic site (8)(9)(10). Cooperativity between active sites is likewise critical for ATP synthesis by F1FO (10,11).To explain both the high degree of cooperativity and the unusual stoichiometry of the enzyme, catalytic mechanisms involving alternating active sites have been proposed (12-14); the models differ in the number of active sites, their order of use, and the involvement of regulatory sites. In the intact ATP synthase, both ATP synthesis and hydrolysis are tightly coupled to proton flow through Fo (15). As Fo appears to contain only one proton channel, consisting in part of the single copy of the a subunit (1, 16-18), only one catalytic site can be coupled and active at one time. Hence any multiplesite mechanism requires a shift in the coupling of Fo to each of the catalytic sites in turn. Such a sequential linking of the proton channel to active sites has been suggested to involve rotation of pa...