Monday, June 06, 2022

Brand insights

In the comments to a previous post met4health provided a link to this paper:

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

Before we can go in to it in depth we have to define a few terms. For this we need Brand's excellent review

Assessing mitochondrial dysfunction in cells

which clarifies many of the terms related to mitochondrial function and their variations between experimental set ups.

Perhaps first term we should look at is the Respiratory Control Ratio (RCR). If you feed a quiescent isolated mitochondrial preparation with substrate but no ADP it consumes a small amount of oxygen. If the preparation is given a briefly supramaximal concentration of ADP (in the presence of permanently high phosphate) there will be a marked rise in O2 consumption while the ADP is converted to ATP. This peak O2 consumption represents the maximum respiration under relatively "physiological" conditions. This is state 3 respiration.

If left alone the ADP is fairly quickly used up and O2 consumption will drop back to basal levels. This level is state 4 respiration.

The RCR is state 3 (maximum possible O2 consumption) divided by state 4 (basal O2 consumption). Conceptually this is a marker of how good a mitochondrial preparation is at upping ATP production when needed. High RCR suggests excellent function.

People have modified the routes to these numbers. The first modification is converting the "idling" state 4 to a "stationary" state. This is done with oligomycin, a complete ATP synthase inhibitor. There are reasons this is done but for now we can just accept it. Brand uses the term state 4oligomycin or state 4o.

Next modification, instead of looking at maximum O2 consumption under surplus ADP, is to simply use a chemical uncoupler to probe maximum possible O2 consumption. This is the peak value of state 3 or state 3 uncoupled (state 3u). If you happen to have used FCCP as your uncoupler you might use the term state 3FCCP.

So RCR is simplified to O2 consumption under FCCP divided by O2 consumption under oligomycin. Crude but effective.

Also Brand says this:

"Net forward flux through each electron transport complex requires a thermodynamic disequilibrium, i.e. the free energy available from electron transfer must be greater than that required to pump protons across the membrane against the pmf."

This translates as: if the proton motive force (pmf) is very high then zero electrons will travel down the ETC, none will reach complex IV and zero O2 will be consumed.

Think about that. Without dissipating a high pmf there is no O2 consumption. This suggests that O2 consumption in state 4oligomycin must be synonymous with uncoupling of various types to allow any O2 consumption at all (necessitating pmf dissipation) in the absence of a functional ATP synthase.

This lets you qualify the information provided by papers which quantify the O2 consumption under oligomycin without mentioning the significance of oxygen being consumed in the absence of a functional ATP synthase.

Brand's review is full of such gems as the above but I think these insights are enough to allow us to go on and look at ROS generation from fatty acid oxidation in the presence/absence of elevated pmf (the electrical component of which is delta psi).


1 comment:

  1. That's a great paper, Peter. Thank you for putting up!