CHEM 445 / BIOL 445
Biochemistry II

J. D. Cronk
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6. header

Lecture 6. ATP synthase & the binding-change mechanism.

Monday 29 January 2007

Energy from electron transport is stored in a proton gradient. Free energy of an electrochemical gradient. Evidence for the chemiosmotic hypothesis. ATP synthase: Structure and mechanism, subcellular localization, subunit composition. Coupling ATP synthesis to proton flux: The binding-change mechanism. Rotational catalysis: ATP hydrolysis also drives rotation in the F1 subunit.

Reading: BTS6, Ch.13, pp.352-353; Ch.18, pp.520-537.

  
6. Summary

Lecture 6 Summary

The chemiosmotic hypothesis: a conceptual breakthrough

The elucidation of the pathway of glycolysis and the means by which the energy of catabolism is coupled to the formation of ATP by substrate-level phosphorylation was a major accomplishment in the history of biochemistry. The mechanism by which the transfer of electrons from NADH to O2 is coupled to ATP generation eluded investigators for several decades, however.

ATP synthase

The transduction of energy of the transmembrane electrochemical gradient back into chemical potential energy (embodied in ATP) is carried out by the ATP synthase complex. The many separate polypeptide chains making up this complex are organized into two major groups. A peripheral assembly with five types of subunits with the stoichiometry a3b3gde carries out ATP synthesis. This is the F1 assembly. An integral membrane assembly called Fo functions as a proton channel.

Binding change mechanism

At the heart of this proposal is the ability of the three b (beta) subunits of the F1 portion of ATP synthase complex to adopt three functionally distinct conformations. The "O" conformation ("open") has very low affinity for the adenine nucleotide substrates (ATP or ADP). The "L" conformation ("loose") has moderate affinity for substrates, but (like the O conformation) is catalytically inactive. Finally, the "T" conformation ("tight") has high substrate affinity and is catalytically active. The catalytic activity of the b subunit is of course the reaction ADP + Pi = ATP + H2O.
Schematic of binding change mechanism
The figure above is an abstract representation of the F1 part of ATP synthase. Following the sequence of events in the figure above from left to right, we see that at the start each the three beta subunits is in a distinct conformation. Thus although the b's are intrinsically equivalent (they are all the same polypeptide chain, and have same general structure) they do not have the same conformation in the vicinity of the active site, and in fact are constrained to be different from one another in the context of the intact ATP synthase holoenzyme. The subunit in the T conformation has a tightly-bound ATP. Addition of ADP + Pi, in the first step in the sequence above, leads to its association with the available catalytic site of the subunit in the L conformation, but no reaction occurs. Now, in the next step, proton flux through the holoenzyme complex drives a conformational change. (We ought to consider how proton flux might be coupled to a conformational change, but for now let us accept that it occurs.) The conformational change converts each of the subunits. The subunit in the T conformation with bound ATP changes to the O form (thus releasing ATP - this key step of ATP release is what distinguishes the behavior of the isolated F1 from that of the holoenzyme in the presence of a transmembrane proton gradient). The subunit in the L form is driven to adopt the T conformation, which then has the ability to catalyze the conversion of the bound ADP + Pi into ATP. The O subunit coverts to the L form, which then has the ability to bind ADP + Pi, and the cycle can be repeated.

We finish our consideration of ATP synthase with the goal of seeing how the Berg-Oster model explains how proton translocation through the F0 subunit is coupled to rotation of the c ring. The latter is linked to the g and e proteins of F1 subunit, so that g rotates to drive conformational changes in the b chains according to the binding change mechanism.

Now, however, we must return to consideration of ATP synthase to ask, "How many protons are required to travel back down the gradient to synthesize an ATP?" Since one rotation of the g subunit of F1 will synthesize three ATP molecules (each of the three b subunits will synthesize one ATP per full 360° g rotation), and given that rotation of g is directly linked to c ring rotation of F0 by the Berg-Oster model, we see that the H+ required to make one ATP depends on the number of c subunits in the c ring. Each c subunit carries one proton around the ring from the cytosolic half-channel to the matrix half-channel of the a subunit. If we assume that the Fo of eukaryotic mitochondria have c rings comprised of 10 c subunits, then its ATP synthase requires 3.33 protons to flow down the potential gradient per ATP synthesized. Putting this together with the analysis in the above paragraph, we see the P:O ratio must be about 3. However, this does not account for the energetic cost of transporting the ATP synthesized in the mitochondrial matrix to the cytosol. Accounting for this makes the P:O ratio closer to 2.5.

Study questions

  • Calculate the free energy of a transmembrane pH difference and/or voltage difference.
  • Describe how the structural model of Fo part of ATP synthase used by Berg and Oster explains how proton flow drives rotation of the g subunit.
  • Sketch a graph of O2 consumption versus ADP supply for an experiment performed on isolated mitochondria, and explain its meaning. Assume the mitochondria have an adequate supply of NADH and Pi.

Page updated 01-06-07

References

  1. Berg, Tymoczko, and Stryer. Biochemistry (BTS): 6th edition (2007, Freeman) Ch.13, pp.352-353; Ch.18, pp.520-527.
  2. Boyer PD. (1997) The ATP synthase-a splendid molecular machine. Ann Rev Biochem 66: 717-749.
  3. Gao YQ, Yang W, Karplus M. (2005) A structure-based model for the synthesis and hydrolysis of ATP by F1-ATPase. Cell 123: 195-202
  4. Noji H, Yasuda R, Yoshida M, Kinosita K. (1997) Direct observation of the rotation of F1-ATPase. Nature 386: 299-302.
  5. Noji H, Yoshida M. (2001) The Rotary Machine in the Cell, ATP Synthase (Minireview) J Biol Chem 276: 1665-1668.
  6. Yasuda R, Noji H, Kinosita K, Yoshida M. (1998). F1-ATPase is a Highly Efficient Molecular Motor that Rotates with Discrete 120° Steps. Cell 93: 1117-1124.
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