© Whole Foods Magazine

September 2002


How Coenzyme Q-10 Works

Part 2: CoQ and Energy Production

An Interview with Dr. Fred L. Crane.

By Richard A. Passwater, Ph.D.

Last month we chatted with Dr. Fred Crane about his discovery of coenzyme Q-10 (CoQ). We learned that it was thinking “outside of the box” that helped lead to this discovery. Dr. Crane, who trained as a plant physiologist, was able to look at mitochondria from animals with a different perspective and then made several startling deductions. In the process, he found a new enzyme, the electron transferring flavoprotein, which helped carry electrons in the mitochondria system and also isolated a quinone-like substance which we now know as CoQ-10 or CoQ. This was extremely startling because at that time, everyone believed that there were no quinones in animals. As Dr. Crane indicated in our last installment, this discovery was not received with ready belief or great joy.

Passwater: Dr. Crane, we left off with your publication identifying CoQ as an indispensable link in energy production. After your publication of this discovery, did things begin to change? Did others join in to follow up or to prove you wrong?

Crane: They mostly try to prove you wrong. Mostly, when you make a discovery everybody tries to prove you wrong. They were busy saying it was an artifact quinone. But when it turned out that it was always there, it was hard to say it was an artifact quinone. The next thing that happened was that it was reduced and oxidized too slowly to function in mitochondria. Mitochondrial cytochromes undergo very rapid oxidation-reduction. Dr. Chance was very much against the concept of CoQ as being functional in mitochondria. He said, “It might be there, but it is not doing anything.”
It took some years later before Dr. M. Klingenberg in Germany was able to show that the kinetics were functioning like a very large pool. In other words, there was a lot of quinone and a few cytochromes, so you didn’t see a one-for-one reduction of quinone and cytochrome. It took 10 cytochromes to get one tenth of the quinone reduced or oxidized. But all that sorted out and eventually everybody came to indicate that CoQ was good for something.

Passwater: Well, I guess it is. Since CoQ is the carrier of electrons between the complexes, doesn’t that make CoQ the limiting factor in this whole electron transport system? If you are short on CoQ, then that’s all the energy you will produce.

Crane: If CoQ is decreased, then the rate of energy conversion is decreased.

Passwater: Of course, you showed that the CoQ in beef heart mitochondria was similar to CoQ from other species.

Crane: Actually, we found five different types of coenzyme Q. We called them coenzyme Q-10 (CoQ), coenzyme Q-9, coenzyme Q-8, coenzyme Q-7 and coenzyme Q-6. For example, rats have coenzyme Q-9, yeast coenzyme Q-6, and humans have coenzyme Q-10 (CoQ). The difference is the number of isoprenoid units in the “tail.” I believe Dr. Karl Folkers and colleagues at Merck were the first to test humans for CoQ.

Passwater: How did CoQ get the alternative name “ubiquinone?”

Crane: That was because of Dr. R. A. Morton. I had read his book to find out how to study quinones. He was a great expert on vitamin A in England. He had found this compound years before we had, and he thought of it in relationship to vitamin A. Then he went along and he looked at it and decided that it was a enedione steroid. That was their hypothesis for what it was when we came out.
 

When Dr. Bob Lester and I at Wisconsin saw it in a spectrum, we said, “Gee, it looks a lot like ours. We’ll write to him and tell him we think it is a quinone.” We were pretty naïve, just a couple of innocent young researchers, and he was a great English biochemistry professor. We assumed there would be no problem writing to an English biochemistry professor and having him discuss something with you. We wrote to him, and he must have gotten a shock because we were saying this was a quinone and was involved in the electron transport in mitochondria. The next thing you knowhe didn’t answer ushe had an article in the British Chemical Journal stating that this compound was ubiquinone. He turned out to be right. It is ubiquitous.

Passwater: He didn’t write back to you and say thanks for the information?

Crane: No, he never wrote back. Actually I talked to him later at some meetings. I can see where he felt pretty bad. Here, he had been working on this thing for some years and it got off on the wrong track. He felt a little disgusted I guess about these hotshots at Wisconsin.

Passwater: He didn’t reference you or anything in the article he published?

Crane: Gosh, I don’t remember. His came out about the same time as ours, so he wouldn’t have been able to reference us.

Passwater: What did you do following the discovery? Did you try to fit more pieces of the puzzle together?

Crane: Our idea was to put the quinone back in. We would do extractions, then put the quinone back in and show that it functioned in restoring activity. We also did fractionations with detergent, got the pieces out and then put the quinone in to show that some of the pieces were quinone reductases or quinone oxidases. In other words, they filled in the gap in the mitochondria. Dr. Joe Hatefi put all the pieces back together with the CoQ and restored the NADH (nicotinamide adenine dinucleotide) oxidation in the mitochondria pieces. Then the pieces were gradually defined as complex 1, 2, 3 and 4. These are shown in the figure on Page XX. Each complex is a cluster of several proteins involved in electron and proton transfer.


Passwater:
This is very difficult for most people to really understand. What you are talking about is essentially taking hydrogen atoms apart into protons and electrons and taking them down this chain of chemicals embedded in the inner membrane of mitochondria.

Crane: It’s like a wire. It is like electricity running down a wire. If you don’t have all the pieces of the wire there, you can’t run the electricity.

Passwater: Or, we can use my analogy of a bucket brigade of molecules carrying electrons. Remember, when bucket brigades were formed in the old days, not all the buckets were identical. This is closer to the situation in the body with the electron transport system the buckets are not identical but consist of about a half dozen different types. If someone in the brigade doesn’t have the correct bucket, then the water won’t get passed along.
What are the key partners for CoQ to do its job?

Crane: CoQ is sort of a connector. In other words, it reacts with dehydrogenases. The dehydrogenases are the succinate dehydrogenase, NADH dehydrogenase, alpha glycero phosphate dehydrogenase, and the fatty acetyl CoA dehydrogenase. All of these feed electrons into the coenzyme pool, and then the reduced CoQ is re-oxidized by the cytochrome bc1 complex. In the special process of NADH CoQ reductase activity, there is a proton movement along with that of the electrons from the inside of the mitochondria to the outside. So that, too, gets involved in energy production.
Then, in the cytochrome bc1 complex, there is a proton and electron movement across the membrane. In other words it is a directed movement. The arrangement of the enzymes in the membrane organizes so that the quinone gets reduced on the one side and oxidized on the other side. It is involved in two sites of proton accumulation on the outside of the membrane.

Passwater: Essentially, electrons and protons move along the mitochondrial membrane in such a way as to build up a charge differentiation across the membrane.

Crane: The electron movement (negative charge) provides energy to pull protons (H+) across the membrane where the positive charge increases.

Passwater: This transport of electrons and protons drives the reaction in which inorganic phosphate combines with adenosine diphosphate (ADP) to make ATP which contains a high-energy phosphate bond that serves as an energy battery. A healthy person should form his or her own weight in ATP daily to supply energy (as the ATP converts into ADP, etc.) for all the reactions needed for a healthy life. Box 2 (see Page XX) shows a schematic of the process.


Crane:
That was the other part of it. Finding out how the electrons moved didn’t tell us how ATP was made. That is where Dr. Mitchell came into the picture. We were dreaming up all kinds of interesting things like the quinone got phosphorylated and then the quinone shifted the phosphate to the ATP none of which worked.

Passwater: But I would think the discovery of two or more of the missing constituents alone would be Nobel Prize worthy. Why weren’t you included as a co-laureate?

Crane: Dr. Mitchell put it into perspective of the membrane in the sense that the electron was moving across the membrane and when it moved across the membrane, it moved charge across the membrane. Therefore, on the other side of the membrane, you got a charge which then accumulated protons. Then the proton concentration on the outside of the membrane would run back in through the ATPase enzyme, and that would be the basis for forming ATP. That is how the ATP formation got finally tied to the whole story.

Passwater: I can see why this was a very complex puzzle to solve. Even after the puzzle has been solved and all the pieces have been elucidated, it still is complex to understand For those interested in overall picture elucidated by Dr. Mitchell, perhaps Box 3 (see Page XX) will be of some help.

Mitchell’s chemiosmotic theory

What Dr. Mitchell proposed was that as hydrogen atoms (i e., electrons and protons) derived from the soluble cytoplasmic substrates of the Kreb’s cycle and fatty acid breakdown are transferred to the mitochondria membrane-associated electron and proton carriers of the respiratory chain, electrons are transferred through the series of carriers. Protons are both utilized and generated during this process asymmetrically with respect to the two sides of the mitochondrial membrane, producing a net result of translocation of protons across the membrane. Thus, a protonic potential difference across the membrane is generated that consists of both an electrical potential arising from the asymmetric charge distribution and a thermodynamic potential arising from the proton concentration differential between the two sides of the membrane. Dr. Mitchell proposed that the potential energy difference and corresponding force associated with this asymmetrical distribution of protons was sufficient to promote the otherwise energetically unfavorable formation of ATP from ADP and inorganic phosphate.


Passwater:
You were the first to isolate CoQ, you identified it and did a lot of research on how it works. Have you ever looked into how CoQ is made in the body?

Crane: No, I have never been involved in that. Dr. Harry Rudney in Cincinnati did a lot of studies. The lipid synthesis people got interested in that and studied the various enzymes involved. They actually sorted out the way it is processed and noted that, genetically, it has been resolved in yeast and e-coli. But that is an area which I didn’t get into.

Passwater: Do CoQ supplements benefit healthy people?

Crane: There are many studies that show improvements in chronic conditions such as heart failure and periodontal disease by supplementation with CoQ.

Passwater: So, in review, the body breaks down very complex chemical structures in food into smaller compounds and even into what are almost the basic elements of physics, electrons and protons, separating them, moving them about and ending up with oxidation products, carbon dioxide and water just as if we were burning the food in air.
This process takes place at a much slower rate, however, because the chemical reactions the transfer of electrons involving oxygen is done step by step instead of all at once.

Crane: It is controlled redox potential change in other words, it occurs in many small steps instead of in one giant leap.

Passwater: Now, thanks to you, we understand how that slow process works.

Crane: Thanks to me and a lot of others.

Passwater: What are your interests today? I notice that you still are giving papers at international symposia.

Crane: I’m interested in the plasma membrane redox system, which is another story. Back in 1972 or 1973, I was working at the Karolinska Institute in Sweden with Dr. Hans Low, who observed that NADH causes a decrease in cyclic-AMP formation. How could that possibly happen? We thought that maybe NADH could cause a reduction in something.
We figured there was a reduction of something in a part of the membrane so I got some plasma membrane at Purdue University, and we assayed it. Goshthere was a very slow oxidation. It was so slow that you had to run the machine for 10 minutes in order to see the change, but it was there.
I said we had some kind of oxidase, and it ended up there was an oxidase, although several laboratories had previously published that there was no redox system in plasma membrane. We had a very difficult time getting people to realize that there was one. We were denounced for all sorts of artifacts.
 

Now, as it turns out, this redox system is somehow involved in the signaling through the membrane because it forms hydrogen peroxide that turns on gene function.

Passwater: This is indeed very interesting. This is the crux of how reactive oxygen species and antioxidant balance regulates gene expression. So far, you have had a very interesting career. Maybe scientists will learn not to be so skeptical when you publish your future discoveries.

 

In the meantime, thank you for your past discoveries, including the discovery of CoQ, and for sharing your story with us. WF

© 2002 Whole Foods Magazine and Richard A. Passwater, Ph.D.

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