Dinitrophenol (DNP) was used in the manufacture of munitions in World War I. In the 1930s, it was used as a weight loss drug. Use in the U.S. cannot be regulated by the FDA because DNP is considered a dietary supplement. Attempts to ban the drug in the U.K. following the death of four users in 2015 failed in Parliament. DNP is a small molecule that is soluble in the mitochondrial inner membrane. The hydroxyl group reversibly dissociates a proton.

A. Predict the effect of DNP on the electrochemical gradient across the inner mitochondrial membrane.

B. Explain how DNP can be used to reduce weight.

 

DNP is an uncoupling agent that affects the proton gradient in the mitochondria during oxidative phosphorylation.

Introduction

Let's talk about the production of the proton gradient.

The electron transport chain (ETC) is made of 4 complexes and the ATP synthase. It is located in the inner membrane of mitochondria:

NADH will unload its electrons in the protein complex #1 while FADH2 will unload its electrons in the protein complex #2. There is a non-protein structure which will move the electrons in complex #1 or #2 to the complex #3 and this structure is called COENZYME Q so, this coenzyme Q is the mobile electron carrier. Then, these electrons should be moved to the complex #4 because this complex is the only one which can interact with oxygen to form H2O...so, we need another carrier and it is called CYTOCHROME C. So, NADH and FADH2 are ELECTRON DONORS while oxygen is ELECTRON ACCEPTOR.

Molecular oxygen is stable but when it interacts with those electrons, it becomes very reactive and negatively charged. Also, from Krebs cycle, there is formation of protons so, these protons will come and interact with the O₂- and form H2O.

When electrons just entered to the electron transport chain, they are very charged but as they pass through all the protein complexes, they lose energy. In complex 1, 3 and 4, as electrons pass through there, they use some energy of these electrons to move the protons from the matrix to the intermembranous space.

So, after this, we will have a high concentration of protons in the intermembranous space and when this happens, protons like to move to the matrix because of electrochemical gradient, it means the intermembranous space will be highly positive and rich in protons levels, while the matrix will have low levels of positive ions and low levels of protons.

The problem is that the inner membrane is not permeable to protons. So, in the inner membrane there is complex #5 and is called ATP SYNTHASE. In this ATP synthase, there is a complex of peptides arranged as a ring with a hole in the center...protons will like to go into this center of the ring and pass to the matrix... When protons pass through this protein complex, this complex starts rotating like a machine. There is another protein attached in the center of this ring which acts as a connector and this is attached to another domain; when this ring starts rotating, it will move the connector domain which will move the last domain and fuse ADP with Pi and form ATP...this last process is called PHOSPHORYLATION.

As inner membrane is impermeable to ATP, then there is a transporter which can move ADP from cytosol to the mitochondrial matrix and ATP from the matrix to the cytosol.

Since the electrons are caught into the electron transport chain until they fuse with O-, this is called OXIDATIVE PROCESS.

All this mechanism of the proton accumulation in the intermembranous space and the ATP synthase, is called CHEMIOSMOTIC HYPOTHESIS OF OXIDATIVE PHOSPHORYLATION.

A. Predict the effect of DNP on the electrochemical gradient across the inner mitochondrial membrane.

DNP is very lipid soluble and it can cross the inner mitochondrial membrane. When it enters the mitochondria, it will go to the intermembranous space, bind protons, cross the inner mitochondrial membrane, and unload the protons in the mitochondrial matrix, it means it will destroy the proton gradient produced during oxidative phosphorylation and as there won't be a proton gradient, then protons won't move from the intermembranous space to the matrix through the ATP synthase (because DNP is doing this job) and there won't be ATP production.

B. Explain how DNP can be used to reduce weight.

DNP can cause a significant increase in the basal metabolic rate. This leads to weight loss by burning more fat and carbohydrates. This occurs because normally, when we eat, our cells use carbohydrates as a source of energy. Remember that carbohydrates are metabolized by aerobic respiration, first glycolysis, then Krebs cycle with the concomitant production of NADH and FADH2 that will enter the electon transport chain to produce ATP by oxidative phosphorylation. When carbohydrates are not available, our cells use FATS as a fuel of energy. As we are blocking the oxidative phosphorylation in the mitochondria by using DNP, then all the NADH and FADH2 produced during carbohydrates metabolism, won't be able to produce ATP by oxidative phosphorylation and as the cells will lack ATP, then the body will start breaking down FATS in order to be used as energy. In this way, the body breaks down fats during the use of DNP, however this can still be dangerous because fats produce ATP by a process called beta oxidation that produces NADH and FADH2, it means fats produce ATP by means of NADH and FADH2 and hence, the oxidative phosphorylation so, even when we are breaking down fats, the energy intermediaries produced by them won't be able to produce too much ATP.

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