Richard L. Cross

Gene duplication as a means for altering H+/ATP ratios during the evolution of Fo F1 ATPases and synthases

In the evolution of the FoF1 family of proton‐translocating membrane complexes, two reversals in function appear to have occurred, first changing it from an ATPase to an ATP synthase and then back again to an ATPase. Here we suggest that with each change in function, the ratio of protons transported per ATP hydrolyzed or synthesized (H+/ATP) was altered in order for the complex to better adapt to its new role. We propose that this was accomplished by gene duplication with partial loss in the number of functional catalytic sites (to increase H+/ATP) or functional proton channels (to decrease H+/ATP). This method of changing the H+/ATP ratio preserved overall structural features of the complex essential to energy coupling.

Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F0F1-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F0 to the endergonic conformational changes in F1 required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the γ subunit of F1 rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F1 and membrane-bound F0F1. Furthermore, the inhibition of this rotation by the modification of F0 with DCCD suggests that rotation in F1 is obligatorily coupled to rotation in F0 as an integral part of the coupling mechanism.

The rotary binding change mechanism of ATP synthases

The F(0)F(1) ATP synthase functions as a rotary motor where subunit rotation driven by a current of protons flowing through F(0) drives the binding changes in F(1) that are required for net ATP synthesis. Recent work that has led to the identification of components of the rotor and stator is reviewed. In addition, a model is proposed to describe the transmission of energy from four proton transport steps to the synthesis of one ATP. Finally, some of the requirements for efficient energy coupling by a rotary binding change mechanism are considered.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

The number of functional catalytic sites on F1-ATPases and the effects of quaternary structural asymmetry on their properties

Recent structural and kinetic studies of F1 and F0F1 are reviewed with regard to their implications for the binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation. It is concluded that at least two and probably all three of the catalytic sites on F1 are functionally equivalent despite permanent structural asymmetry in the soluble enzyme. A rotary mechanism in which all three catalytic subunits experience all possible interactions with the single-copy subunits during turnover is thought not to apply to soluble F1 but remains an attractive model for the membrane bound enzyme.

ATP hydrolysis by membrane-bound Escherichia coli F0F1 causes rotation of the γ subunit relative to the β subunits

We recently demonstrated that the gamma subunit in soluble F1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10964-10968). Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex. It is shown that beta D380C-F1, containing a beta-gamma intersubunit disulfide bond, can bind to F1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution approach with crosslinked beta D380C-F1, beta subunits containing an N-terminal Flag epitope (beta flag) were incorporated into the two non-crosslinked beta positions and the hybrid F1 was reconstituted with membrane-bound F0. Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta flag to the gamma subunit. This demonstrates that gamma rotates within F1 during catalytic turnover by membrane-bound F0-F1. Furthermore, the rotation of gamma is functionally coupled to F0, since preincubation with DCCD to modify F0 blocked rotation.

Rotation of the ε Subunit during Catalysis by Escherichia coli FOF1-ATP Synthase

We report evidence for catalysis-dependent rotation of the single ε subunit relative to the three catalytic β subunits of functionally coupled, membrane-bound FOF1-ATP synthase. Cysteines substituted at β380 and ε108 allowed rapid formation of a specific β-ε disulfide cross-link upon oxidation. Consistent with a need for ε to rotate during catalysis, tethering ε to one of the β subunits resulted in the inhibition of both ATP synthesis and hydrolysis. These activities were fully restored upon reduction of the β-ε cross-link. As a more critical test for rotation, a subunit dissociation/reassociation procedure was used to prepare a β-ε cross-linked hybrid F1 having epitope-tagged βD380C subunits (βflag) exclusively in the two noncross-linked positions. This allowed the β subunit originally aligned with ε to form the cross-link to be distinguished from the other two βs. The cross-linked hybrid was reconstituted with FO in F1-depleted membranes. After reduction of the β-ε cross-link and a brief period of catalytic turnover, reoxidation resulted in a significant amount of βflag in the β-ε cross-linked product. In contrast, exposure to ligands that bind to the catalytic site but do not allow catalysis resulted in the subsequent cross-linking of ε to the original untagged β. Furthermore, catalysis-dependent rotation of ε was prevented by prior treatment of membranes with N,N′-dicyclohexylcarbodiimide to block proton translocation through FO. From these results, we conclude that ε is part of the rotor that couples proton transport to ATP synthesis.

Binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation

Publisher Summary This chapter discusses binding change mechanism for adenosine triphosphate (ATP) synthesis by oxidative phosphorylation and photophosphorylation. It presents a three-site version of the binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation. The chapter presents some experiments, the results of which not only clearly demonstrate the existence of the high-affinity site during steady-state catalysis, but the excellent agreement of the data with the calculated plot also provides an independent confirmation of published values for k 1 and k 3 . The effect of an ATP-regenerating system on catalytic site occupancy at low ATP concentration is also presented. F 1 was incubated with [ 3 H] ATP long enough to allow approximately 10 turnovers per F 1 . Samples were removed to determine the amount of nucleotide bound to F 1 and the total [ 3 H] ADP in the reaction mixture. Increasing concentrations of pyruvate kinase decrease medium adenosine diphosphate (ADP) significantly but they have little effect on catalytic site occupancy as shown by the amount of tritiated nucleotide retained by F 1 upon removal of unbound ligand. These results demonstrate that medium ADP is not required for observing the high-affinity site. Another approach to measure the equilibrium constant for ATP synthesis at the catalytic site in the absence of energization is to determine the amount of bound [ 32 P]ATP formed from the phosphorylation of bound ADP by medium 32 P i . The dependency of the size of this ATP fraction on the P i concentration suggests that under these conditions, the Κ D for the P i binding to a catalytic site containing ADP might be in excess of 0.1 M .

A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure

An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and theras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the β subunit of F1. Several potentially important residues on β are noted that have not yet been studied by mutagenesis or affinity labeling.

Chapter 13 The reaction mechanism of F0F1ATP synthases

Publisher Summary This chapter discusses the reaction mechanism of F0F1-ATP Synthases. ATP synthesis by oxidative phosphorylation and photophosphorylation is catalyzed by a complex of proteins, F0F1, found embedded in the membranes of eubacteria (BF0F1), mitochondria (MF0F1), and chloroplasts (CF0F1). The main focus of this chapter is on the advances in understanding of the mechanism of ATP synthesis by F0F1. Since the development of mechanistic models, there has been strong influence in the advances in knowledge of structure, a brief review of structural features of the complex precedes discussion of mechanism. The number of unmodified catalytic sites would have to be reduced to one in order to eliminate residual bi-site catalysis, and the stoichiometry for inhibition of multi-site catalysis would be two mol/mol. Another type of probe may exist that partially impairs cooperative interactions at unmodified sites when incorporated into a single site. A third class of affinity probe may exist which, when incorporated into one site, freezes the enzymes conformation in such a way that promoted catalysis at the remaining two sites is fully blocked.