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

Abstract

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...

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

“…We and others have earlier shown that αSer344 in the α 341 NVISIT 346 helix plays an important role in the preparation of the transition state (TS) (15,31). Our QM results have also suggested that the side chain of αS344 has to turn to the active site and form a hydrogen bond with O=γP of ATP (15). This flipping of the Ser side chain can be observed in the MD simulation in T* after ∼9.7 ns (Fig.…”

“…According to single-molecule experiments, the bond-breaking step takes place after an ∼90°rotation of the γ-subunit, thus in between the two conformations, most likely in a near-D structure (12). This conclusion was supported by theoretical studies (13,15,16), and recently, we provided indications that the barrier height for the hydrolysis step decreases significantly when the active site conformation changes from T to D (15,16). Additional support was obtained from single-molecule manipulations (17).…”

“…However, all of the interresidue distances eventually spontaneously decrease to the distance values observed in D, enhanced also by a contribution from αS344 forming C2. We and others have earlier shown that αSer344 in the α 341 NVISIT 346 helix plays an important role in the preparation of the transition state (TS) (15,31). Our QM results have also suggested that the side chain of αS344 has to turn to the active site and form a hydrogen bond with O=γP of ATP (15).…”

“…When T and D were superposed, the largest change in the active site is the ∼1.1-Å shift of the αR373 residue (7). This residue has earlier been shown important for the reaction (5,15,19), although how its position may constitute a control mechanism is not understood; also, it is not understood how long-range interactions may connect the conformation at the reaction center to the rotary position of the γ-subunit. …”

Why nature really chose phosphate

“…Obviously, this is still a work in progress ; however, it also illustrates the need for careful experimental and theoretical analyses of this problem. In this respect, it may be useful to expand here on current QM/MM studies of ATP hydrolysis in proteins and in solution (Akola & Jones, 2006 ;Beke-Somfai et al 2011 ;Dittrich et al 2003 ;Grigorenko et al 2007b ;Li & Cui, 2004), some of which have not attempted to obtain quantitative results (e.g., Dittrich et al 2003), and others of which have major problems due to not having included a sufficiently large part of the environment, and in having no sampling. In fact, the difficulties of reproducing the observed barrier led to the problematic message that has emerged from the work of (Grigorenko et al 2007b).…”