ATP Synthesis: -Chemiosmotic Coupling Allows Nonintegral Stoichiometries of O2 Consumption and ATP Synthesis
Before the general acceptance of the chemiosmotic model for oxidative phosphorylation, the assumption was that the overall reaction equation would take the following form:
with the value of x—sometimes called the P/O ratio or the P/2e- ratio—always an integer. When intact mitochondria are suspended in solution with an oxidizable substrate such as succinate or NADH and are provided with O2, ATP synthesis is readily measurable, as is the decrease in O2. Measurement of P/O, however, is com plicated by the fact that intact mitochondria consume ATP in many reactions taking place in the matrix, and they consume O2 for purposes other than oxidative phosphorylation. Most experiments have yielded P/O (ATP to 1/2 O2) ratios of between 2 and 3 when NADH was the electron donor, and between 1 and 2 when succinate was the donor. Given the assumption that P/O should have an integral value, most experimenters agreed that the P/O ratios must be 3 for NADH and 2 for succinate, and for years those values have appeared in research papers and textbooks.
With introduction of the chemiosmotic paradigm for coupling ATP synthesis to electron transfer, there was no theoretical requirement for P/O to be integral. The relevant questions about stoichiometry became, how many protons are pumped outward by electron transfer from one NADH to O2, and how many protons must flow inward through the FoF1 complex to drive the synthesis of one ATP? The measurement of proton fluxes is technically complicated; the investigator must take into account the buffering capacity of mitochondria, nonproductive leakage of protons across the inner mem brane, and use of the proton gradient for functions other than ATP synthesis, such as driving the transport of substrates across the inner mitochondrial membrane (described below). The consensus values for number of pro tons pumped out per pair of electrons are 10 for NADH and 6 for succinate. The most widely accepted experi mental value for number of protons required to drive the synthesis of an ATP molecule is 4, of which 1 is used in transporting Pi, ATP, and ADP across the mitochondrial membrane (see below). If 10 protons are pumped out per NADH and 4 must flow in to produce 1 ATP, the proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate. We use the P/O values of 2.5 and 1.5 throughout this book, but the values 3.0 and 2.0 are still common in the biochemical litera ture. The final word on proton stoichiometry will prob ably not be written until we know the full details of the FoF1 reaction mechanism.
FIGURE 19–25 Rotation of Fo and experimentally demonstrated. F1 genetically engineered to contain a run of His residues adheres tightly to a microscope slide coated with a Ni complex; biotin is covalently attached to a c subunit of Fo. The protein avidin, which binds biotin very tightly, is covalently attached to long filaments of actin labeled with a fluorescent probe. Biotin-avidin binding now attaches the actin filaments to the c subunit. When ATP is provided as substrate for the ATPase activity of F1, the labeled filament is seen to rotate continuously in one direction, proving that the Fo cylinder of c subunits rotates. In another experiment, a fluorescent actin filament was attached directly to the γ subunit. The series of fluorescence micrographs shows the position of the actin filament at intervals of 133 ms. Note that as the filament rotates, it makes a discrete jump about every eleventh frame. Presumably the cylinder and shaft move as one unit.
