Oxidative Phosphorylation Inhibition, Uncoupling, and ROS

How do Complex I inhibitors work?

They inhibit the oxidation of Fe-S clusters and prevent transfer of electrons to CoQ. NADH cannot transfer electrons to CoQ and no protons are pumped.

Complex I inhibitor examples

Rotenone, Demerol, and Amytal

How do Complex III inhibitors work?

They inhibit the transfer of electrons from Cyt b to CoQ. This also increases ROS production, which is toxic to cells.

Complex III inhibitor examples

Antimycin A

How do Complex IV inhibitors work?

They bind to the iron centers of heme a3 and prevent the transfer of electrons from Cyt C to O2 (H2O).

Complex IV inhibitor examples

Cyanide, Azide, and Carbon Monoxide

How do ATP Synthase inhibitors work?

They block the movement of H+ through F0 by binding to C10 and preventing rotation. Without proton flow, no conformational change occurs and ATP is not synthesized.

ATP Synthase inhibitor examples

Oligomycin

How can the ETC and ATP Synthesis become unlinked?

If the protons can find an alternate pathway to dissipate out of the intermembrane space and into the matrix, ATP synthesis will not occur but the ETC can continue to work because there is no buildup of protons that prevents flux through the ETC. The energy stored in the proton gradient is released as heat.

What molecules or proteins can cause uncoupling?

2,4-Dinitrophenol (DNP) and Uncoupling Proteins (UCPs)

How does 2,4-Dinitrophenol (DNP) cause uncoupling?

It acts as an ionophore that can pick up protons in the intermembrane space (high pH = protonation) and diffuse across the membrane, dropping off the protons in the matrix (low pH = deprotonation).

How do uncoupling proteins (UCPs) cause uncoupling?

They provide a transport channel that allow protons to flow from the intermembrane space into the matrix. UCPs are expressed in many cells and actually play an important role in thermogenesis (useful for hypothermia and hibernation) and protecting against ROS generation.

Uncoupling Protein example

UCP-1 Thermogenin is expressed in brown adipose tissues (BATs) and plays a role in nonshivering thermogenesis. It metabolizes fats to generate heat, and is used by hibernating mammals to produce body heat and maintain organ function.

Endogenous ROS

ROS that are synthesized in the body, such as those that result from normal ETC function.

Exogenous ROS

ROS that come from outside of the body, such as from pollutants, tobacco, smoke, drugs, and radiation.

3 important antioxidants

Superoxide dismutase, glutathione peroxidase, and catalase

Superoxide Dismutase

The first line of defense against ROS. Can generate O2 (safe) or H202 (hydrogen peroxide; unsafe, must be processed by another enzyme or can become a toxic hydroxyl radical through Fenton chemistry).

Glutathione Peroxidase

Oxidizes Glutathione (GSH) to GSSG in order to reduce H2O2 to H2O. GSSG is reduced back to GSH by Glutathione Reductase and NADPH. This process mostly occurs in erythrocytes and relies on the PPP.

Catalase

Uses Fe ions to reduce H2O2 to H2O and O2. Highly efficient enzyme that protects the mitochondria from oxidative stress.

Superoxide

The precursor for all ROS.

Hydroxyl radical

The most potent ROS. Formed by Fenton chemistry (oxidation of Fe splits hydrogen peroxide).