Lecture 7 Summary

The overall rate of electron transfer and its consequent consumption of oxygen depends on the availability of ADP. The nature of this so-called respiratory control insures that ATP is generated when needed. Various inhibitors of electron transport help reveal the sequence of electron carriers. Experimentally, the operation of the ETC is indicated by the consumption of oxygen (i.e. its reduction to water) by a suspension of mitochondria provided with a suitable reductant (source of electrons). The concentration of oxygen can be monitored by an oxygen electrode. When such an experiment is carried out, it is found that electron transport ceases even in the plentiful presence of NADH when the supply of ADP is exhausted.

Similar experiments show that the block of the ETC by inhibitors amytal or rotenone can be bypassed by adding succinate to the mitochondrial suspension. This shows that these inhibitors prevent the flow of electrons through complex I. The blockage due to another inhibitor, antimycin A, cannot be relieved by succinate addition. However, addition of the nonphysiological reductant TMPD, along with ascorbate, allows oxygen consumption to resume. This is because TMPD is an ascorbate-reducible redox carrier that can transfer electrons directly to cytochrome c. This shows that antimycin A interferes with electron transfer through complex III. Finally, the inhibitors cyanide, azide, and carbon monoxide block electron transport at the heme a3-CuB site in complex IV. Since this is the site at which oxygen is reduced, there is no bypass electron donor. The failure to find any such electron donor would suggest that these inhibitors act very late in the ETC.

Time-resolved spectroscopic experiments are complementary to those described above (more details to be posted later).

Another feature of oxidative phosphorylation that is useful for us to consider is the amount of ATP synthesized per oxygen atom reduced, the so-called P:O ratio. The three free energy gaps between NADH, coenzyme Q, cytochrome c, and oxygen each appear to adequate to drive the synthesis of one ATP. Of course, we know that ATP is not directly synthesized by the exergonic electron transfers, but instead is used by Complexes I, III, and IV for proton translocation to generate a transmembrane electrochemical gradient. Our text (BTS, p.517) suggests that for each pair of electrons that flow through the ETC, the best current "estimates" for the number of protons moved from inside to outside by these complexes are 4, 2, and 4, respectively, for a total of 10. Note that a pair of electrons is required to reduce one electron, so the "H+ translocated:O" ratio is 10 (for electrons donated to the ETC by NADH).

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

• 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 P_i.

Page updated 01-06-07

References

1. Boyer PD. (1997) The ATP synthase-a splendid molecular machine. Ann Rev Biochem 66: 717-749.
2. 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