May 22, 2019 | Ross Anderson

Automatic Emergency Brake Found on Molecular Motor

In this article, guest writer Ross Anderson (PhD, biochemistry professor) discusses a new finding about how an ’emergency brake’ works in one of the most vital of all molecular machines, ATP synthase. First, he explains the molecular rotary motor and what it does.

Review of “Extrinsic conditions influence the self-association and structure of IF1, the regulatory protein of mitochondrial ATP synthase” by Boreikaite et al., PNAS.

Evaluated by Ross S. Anderson, Ph.D.

First let’s take a brief look at what the ATP synthase (here referred to simply as the Synthase) is and its importance to life. In eukaryotes the Synthase is located in the mitochondrial inner membrane. In prokaryotes, such as E. coli, it is located in the cell membrane as these organisms lack mitochondria.

Why ATP Is Important

Adenosine triphosphate (ATP) is one of several high-energy compounds used by all cells to drive reactions necessary for life. Without it, life as we know it would not be possible. While it is not the only high-energy compound in the cell, it is the one most widely used by cells. To carry out its moment-to-moment activities a cell must have a constant supply of ATP. Most of the cell’s ATP needs are supplied by the ATP synthase molecular motor.

In eukaryotes, such as humans, the mitochondria are the “power houses” of the cell as these contain the Synthase. In order for the synthase to synthesize ATP, there must be a steady supply of oxygen. Oxygen is delivered to the cells via hemoglobin in the red blood cells. The oxygen is then transferred to another protein in the cell known as myoglobin (responsible for the red color of muscle), myoglobin then carries the oxygen to the mitochondria where it serves as an electron acceptor to form water. The electrons come from food molecules eaten by the organism. They are removed by a variety of metabolic pathways and given to electron-carrier molecules such as NAD+ and FAD to form NADH and FADH2, respectively.

Optimizing the Motors

Electrons from FADH2 and NADH are then transferred to protein complexes also located in the inner mitochondrial membrane. As the electrons are passed from one protein complex to another and ultimately to oxygen, they are used to power what are termed proton “pumps” which pump protons (H+) out of the mitochondrial matrix (the inner most part of the mitochondria) to the intermembrane space (the space between the inner and outer membranes of the mitochondria). This sets up a proton gradient with a high concentration of protons outside (intermembrane space) and a lower concentration inside (matrix).

As protons travel back into the matrix down the gradient, the potential energy in the gradient is used to synthesize ATP. The inner membrane is impermeable to the protons, so to reach the matrix, the protons must travel through the Synthase. As they travel through the synthase, they cause an internal rotor, called the γ subunit to rotate in increments of 120°. As the γ subunit rotates, its contacts with the α3β3 subunits that comprise the knob part of the Synthase protruding into the matrix, cause a series of conformational changes in the 3 β subunits leading to the binding of ADP + Pi, the synthesis of ATP, and the release of ATP.

The Role of Oxygen

As stated above, in order for the synthase to synthesize ATP, there must be an ample amount of oxygen. However, if the blood supply to an organ is blocked, ischemia, then oxygen delivery is compromised. Under such hypoxic conditions, the Synthase would tend to rotate in the reverse direction and degrade precious ATP. For cells of the heart, the brain and other highly aerobic organs this can be life-threatening. To prevent this from happening, a small polypeptide of variable length, depending on the organism, has been designed to become active and bind to the α3β3 and γ subunits in such a way as to prevent the γ subunit from rotating in reverse and consuming ATP. This polypeptide is called IF1 (inhibitory factor 1).

Mitochondrial membranes are optimized to concentrate protons where the ATP synthase motors are. They, in turn, are aligned in pairs to optimize ATP production.

An Automatic Brake

Under hypoxic conditions (low oxygen), the pH of the cell decreases (becomes more acidic), and this causes the IF1 to form dimers which are active and bind to the F1 region of two adjacent Synthases and cross-link them. There is a modest investment of 2 ATP consumed per binding of an IF1, but the savings in ATP can be significant. If conditions return to normal, and the pH rises again, the IF1 dimers form inactive tetramers and higher oligomers. One might compare it to the “hill stopper” function found in many newer cars. This mechanism engages the brakes if it senses that the vehicle is starting to roll backwards down a hill. It stops the wheels, allowing the driver to shift gears without fear of rolling into something behind them.

Interestingly, under hypoxic conditions the E coli Synthase is allowed to run in reverse and consume ATP to pump H+s out of the cell; i.e., there is not an IF1-like polypeptide. This is because bacteria use proton gradients for many other purposes, not just the making of ATP. Even under hypoxic conditions the maintenance of these gradients is essential. The bacteria can increase their yield of ATP under these conditions by greatly increasing their consumption of glucose, as ATP can be generated from glucose via the glycolytic pathway which does not require oxygen.

The energy demands of eukaryotic cells are greater than prokaryotic cells. Consequently, it is imperative that their mitochondria be supplied with ample amounts of oxygen in order generate the necessary quantities of ATP. While eukaryotic cells also have pathways to generate ATP that do not require oxygen; e.g., glycolysis, these cannot generate the sustained quantities of ATP needed. Any situation that would lead to the consumption of ATP unnecessarily without getting useful work out of it would quickly threaten the life of the cell. Anyone can recognize the “hill stopper” function used in many cars is obviously a designed feature. Similarly, the IF1 is also a design feature in living things used to prevent the unwanted consumption of ATP. It had to be in place and fully functional with the first eukaryotic cells.

Good Science Doesn’t Need Evolutionary Speculation

Now that we’ve briefly looked at the Synthase and IF1, and their roles in the cell, let’s look at the article in PNAS.

This paper is a good example of scientific research carried out and reported without invoking an evolutionary perspective. No doubt the authors believe in evolution, but they refrained from mentioning it here. This illustrates that important research can be done without a belief in evolution to guide it as nowhere in the article did the authors refer to evolution as a guiding principle. Indeed, no mention of evolution was made.

Science, as illustrated by this article, is well equipped to address three questions:

  • What is it? The answer to this question is to provide merely descriptive details as to what it looks like, what kind of molecule is it etc. These authors answered this question quite easily in describing the structure of the bovine IF1; an 84 amino acid polypeptide that binds to the F1 region of ATP synthase and inhibits it.
  • What does it do? This question addresses the function of the object of study. This question was also easily addressed by the authors which explained how this polypeptide binds to the F1 portion of the ATP synthase and how this leads to its inhibition. They discussed how pH, protein concentration and Ca2+ concentration influence the formation of active dimers from inactive tetramers.
  • How does it do what it does? This question asks how the object carries out its function. This question, too, was addressed by the authors. Under low oxygen conditions, such as hypoxia due to ischemia, the pH in the mitochondria decrease, the electron carriers in the inner membrane of the mitochondria are no longer able to pass the electrons onto oxygen and thus become progressively reduced. This leads to a collapse of the proton motive force used to power the synthesis of ATP by the Synthase. Under these conditions, the Synthase tends to do the reverse reaction and degrade ATP to ADP and Pi, thus quickly depleting the ATP concentration of the cell. To prevent this from happening, the lowered pH destabilizes the tetrameric complex of IF1 causing it to be dismantled into the active dimeric form. The dimeric form binds to the F1 portion of the Synthase and interacts with both the α and β subunits as well as the γ Interaction with the γ subunit inhibits its rotation and thus prevents the Synthase from working in reverse to destroy ATP.

All too often authors of books and journal articles introduce a fourth question into the mix after addressing the first three in some detail. The fourth question is: How did it come to be? Detailed answering of the first three questions leads the reader to unwittingly have confidence in the answers to this fourth question; they assume that the author(s) know what they are talking about and uncritically accept their answers. What many fail to understand is that in addressing the fourth question one steps out of the arena of science and into the arena of philosophy or worldview. To their credit these authors did not do that, but instead they treated the data and its interpretation as they should have without trying to prop up a belief in evolution.

Dr. Anderson’s expertise is in the area of biochemistry and molecular biology. He has taught Biochemistry and helped to direct research projects of graduate and medical students at Baylor College of Medicine, Houston, TX. Dr. Anderson was a post-doctoral researcher in the Molecular Genetics Division of the Department of Ophthalmology at the Houston Neurosensory Center.

Dr. Anderson was a member of both the undergraduate and graduate faculty at Lamar University, Beaumont, TX. There he taught and directed the research activities of undergraduates and Masters of Science degree candidates in Biology. Currently he is professor of biochemistry at The Master’s University in southern California.

Dr. Anderson’s research interests include structure-function studies of DNA polymerizing enzymes and the synthesis and expression of synthetic human genes in bacterial hosts. He has authored or co-authored several publications in major, peer-reviewed journals. He is a member of the American Chemical Society and Sigma Xi Research Society.

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  • Andrei says:

    Excellent breakdown of the material and summary of the PNAS article. Thank you, Dr. Anderson.

    If possible, I’d like to pose a question that I’ve been wondering about for some time. It was partly brought up during the initial discussion of how ATP is formed, particularly under hypoxic conditions. We know that aerobic respiration produces ATP a lot more efficiently than anaerobic respiration; my question is whether or not it could be feasible to attempt a calculation of ATP synthesis rate under anaerobic conditions in order to deduce how much physical time would be required by a bacterium to produce its essential proteins.

    For example, in my old Biochem text we had a formula to describe how much ATP is needed for a protein of a certain size (eg, amino acid sequence) to be made. The formula was “(4n)-1, where n=number of amino acids.”

    We were told aerobic glycolysis is roughly 38% efficient, creating 36 ATPs from one glucose molecule, and only about 3-5% efficient using anaerobic glycolysis to make just 2 ATPs from one glucose.

    We then calculated the rate of ATP synthesis and determined that, for anaerobic glycolysis, roughly 2 ATPs are produced every ~30 seconds. Does this sound accurate?

    If so, we can then try to calculate roughly how many ATPs are needed to produce an average number of proteins in a hypothetical small anaerobic bacterium (roughly ~1,000 proteins last time I searched), the average length of those proteins being roughly 300 amino acids. Using the above formula, the number of ATPs needed in this case would 4(300,000)-1 = 1,199,999. Does this number seem accurate?

    If it takes about 30 seconds to produce 2 ATPs, and we need nearly 1.2 million of these molecules, that means if we have 240 ATPs/Hr and 5760 ATPs/day, then 1,999,999 ATPs/5760 gives us 208.3 days needed. I’d appreciate a numbers check so far.

    If the above is reasonable, then we have to conclude that a small anaerobe containing about 1000 proteins, each about 300 a.a. long would require nearly 7 months to synthesize all the ATPs it needs for these production of these proteins. Obviously we are not taking into account other ATP requirements of that anaerobe’s life functions such as communication, mobility, etc., only the energy needed to produce those essential 1000 proteins.

    Summing up, and back to my question: given the above, it leads me to conclude that if it takes over half a year to produce these proteins, what does this do to the replication cycle of the anaerobe? Would we expect to see barely 2 anaerobes capable of producing their proteins per year?

    • Andre,

      Thanks for your comment. Your calculations appear to be correct. Though I believe that the efficiency of the Synthase has been upped from the 38% to >60%, even as high as 90% by some estimates! However, even with these higher estimates I don’t see that it will solve the problems you raise.

      You do raise some interesting questions that need further inquiry. Under stressful conditions, such as hypoxia, the entire metabolic activity of cells, pro- and eukaryote, will be altered so as to use much less energy. How long that can be sustained? I don’t know—good to look at. Essentially, the increase in ATP production via glycolysis coupled with lower metabolic activity, the ATP made can be used to power the FoF1 ATP synthase in reverse to maintain a H+ gradient which is used to drive many other processes necessary to sustain life of the bacterium.

      Clearly, the impact this would have on the evolution of cells is enormous as it clearly suggests the impossibility of evolution of cells living under hypoxic conditions as proposed for the early earth.

      Again, thanks for your comments and thought-provoking questions.


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