Energy Coupling in Cation-Pumping Pyrophosphatase—Back to Mitchell

摘要

“Scientists frequently debate theories”. Douglas Allchin Introduction Those of a certain age may remember (and their younger colleagues can read) accounts of the vivid debate in the 1970s surrounding the coupling mechanism involved in oxidative and photo phosphorylation. By that time, Mitchell's chemiosmotic hypothesis had already gained credence, and the debated issue was how a transmembrane H ~(+) potential difference drives ATP synthesis by F-type ATP synthases. The major mechanisms that were considered assumed that the membrane (F _(o)) and peripheral (F _(1)) parts were functionally connected in different ways. Peter Mitchell proposed a “direct coupling” mechanism in which protons are translocated through F _(o) into the catalytic site of F _(1), where they participate directly in ADP phosphorylation and form water as the second product ( Mitchell, 1974 ). Paul Boyer, the proponent of the main competing mechanism, advocated an “indirect coupling” mechanism (successively termed “alternating site”, “binding change”, or “rotational”) that implied that protons transfer their energy to the catalytic site indirectly, via distant conformational strain ( Boyer, 1997 ). The debate was resolved in favor of Boyer's mechanism when it became clear that the alternative mechanism is inconsistent with H ~(+)/ATP stoichiometry and, finally, when the three-dimensional structure of the F-ATPase was determined ( Abrahams et?al., 1994 ). Now, after several decades, the problem of energy coupling is being revisited in connection with membrane pyrophosphatases (mPPases), ancient transporters that couple H ~(+) and Na ~(+) transport across biological membranes in plant vacuoles and bacteria to pyrophosphate hydrolysis. mPPases are functional analogs of F-type ATPases and similarly catalyze a direct attack of a water molecule on a phosphorus atom without formation of a phosphorylated intermediate. However, mPPases have a much simpler structure; each of the two identical subunits of mPPase consists of 15?17 transmembrane α-helices, and six of them form the catalytic site on the cytosolic side. H ~(+)-transporting mPPases (H ~(+)-PPases) have been known since 1966 ( Baltscheffsky et?al., 1966 ; Serrano et?al., 2007 ) and are recognized as contributors to plant stress resistance ( Yang et?al., 2014 ). More recent studies have identified an evolutionarily related prokaryotic Na ~(+)-transporting mPPase lineage (Na ~(+)-PPases) that can pump both H ~(+) and Na ~(+) ( Malinen et?al., 2007 ; Luoto et?al., 2013a ; Luoto et?al., 2013b ). mPPase studies have been further boosted by publication in 2012 of the three-dimensional structures of the H ~(+)-transporting mPPase from Vigna radiata ( Lin et?al., 2012 ) ( Figure 1A ) and the Na ~(+)-transporting mPPase from Thermotoga maritima ( Kellosalo et?al., 2012 ). Two mechanisms to explain coupling between PP _(i) hydrolysis and H ~(+) (Na ~(+)) pumping, proposed based on these structures, differ principally in the order of hydrolysis and transport events and the role of the proton released by the attacking water nucleophile. Figure 1 Membrane pyrophosphatase as an H ~(+) and Na ~(+) transporter. (A) Two views of a subunit of V. radiata homodimeric H ~(+)-pyrophosphatase, showing elements of the transport machinery [PDB code: 4A01; Lin et?al., 2012 )]. The image on the right is a top view from the cytosolic side. Blue sticks, imidodiphosphate; red sphere, water nucleophile (the oxygen atom); green spheres, three gate-forming residues (Arg242, Asp294, and Lys 742); imidodiphosphate-liganded Mg ~(2+), and K ~(+) ions are not shown. Created with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4, Schrodinger, LLC). (B) Mitchell-type coupling of PP _(i) hydrolysis with H ~(+) transport in a single subunit. The ions (atoms) directly involved in the transport process are marked by colored circles. Two aspartate residues (Asp287 and Asp731 in V. radiata mPPase) coordinate and activate the nucleophilic water molecule during its attack on PP _(i). (C) Electrometric traces of V. radiata pyrophosphatase-loaded liposomes obtained with a Nanion SURFE ~(2)R N1 instrument. Currents were recorded following the addition of K _(4)PP _(i), methylene diphosphonate (MEDP), and K _(2)HPO _(4) in the absence and presence of the protonophore CCCP (carbonyl cyanide m -chlorophenyl hydrazone). This panel was reproduced with permission from Shah et?al. (2017) . (D) A billiard-type mechanism of Na ~(+) transport at a low Na ~(+) concentration. The nucleophile-generated H ~(+) pushes out the gate-bound Na ~(+) (coordinated by Asp243, Glu246, and Asp703 carboxylates in T. maritima mPPase; Li et?al., 2016 ) and passes the gate itself in the same or successive turnover. (E) Inhibition of Na ~(+) transport by a Na ~(+) ion bound at a low-affinity transitory site N. The identities of the residues forming it are yet unknown. (F) An alternative mechanism of concurrent Na ~(+) and H ~(+) transport by different subuni