Complex IV and control of delta psi

There was a time, quite early in my anaesthesia training, when we used to use a calcium infusion to support blood pressure in anaesthetised horses. You got a bottle of calcium borogluconate marketed for treating milk fever in cattle, hooked it up to a giving set and chose a ball park drip rate by eye. It was bloody effective, easy to use and dirt cheap.

Then we learned a bit more about the role of Ca2+ in cell death and stopped doing it. It's still worth thinking about why it worked.

I have accepted various concepts about the acute control of delta psi and ROS production when metabolic substrate is supplied in excess of metabolic needs. The basic idea is that a replete ATP pool allows delta psi to rise and generate ROS. The earliest ref I've got in support of delta psi and ROS comes from Skulachev in the late 1990s.

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

This is not a physiological model, it just looks at how manipulating delta psi with substrate/inhibitors controls ROS generation. Peak delta psi specified on the graph with their voltage sensitive dye appears to be around 170mV.

The peak values of delta psi and ROS generation are under succinate oxidation and delta psi is modified using either an uncoupler or complex II inhibitor, so, as so often, we are a long way from physiology here but the general principle that ROS generation rises rapidly above a threshold delta psi appears to hold good today. Currently the rise in ROS is thought to occur at around 140mV. Next we can think about the control of ATP synthesis by complex IV, synonymous with cytochrome c oxidase.

This is an interesting review/hypothesis paper from 2001 but I think it too is now quite well accepted:

New Control of Mitochondrial Membrane Potential and ROS Formation – A Hypothesis

Peter Mitchell's original concept, to which I have long-term "subscribed", was that electrons passed down the ETC to oxygen, generating a proton gradient, which generates ATP via ATP synthase. If the proton gradient becomes high enough it is no longer possible for electrons to force the extrusion of any more protons (or to be able to flow down the ETC to oxygen) so respiration slows. This appears to be real and to happen at a membrane voltage of 140-200mV. I've extracted the two components of Figure 4 in to separate graphs for a clearer discussion.

Like this:

The red line is the rate of respiration through complex IV as a function of the delta psi generated. As the membrane voltage increases ATP synthase starts turning at around 60mV (the blue line). At just over 100mV ATP synthase activity is maximal and doesn't increase with increasing membrane voltage (in this model). What does increase are those aforementioned ROS generated above 140mV. 

Summary so far: The very high membrane voltages needed to inhibit respiration at complex IV will cause excess ROS generation. This is on the border between physiology and pathology.

There is a second system to control respiration through complex IV. This system monitors the ATP:ADP ratio and limits respiration (and membrane voltage to minimal ROS generating levels) based on rising ATP levels. Like this:

The blue line of ATP synthase activity is unchanged. The green line of respiration though complex IV, as soon as ATP synthase starts to generate ATP, begins to drop and limits respiration though complex IV with a maximum membrane potential at around 120mV, well below that 140mV needed for ROS generation.

So we can limit respiration by inhibiting complex IV using this system at a membrane potential below 140mV with limited ROS generation or we can inhibit it at above 140mV accepting ROS generation using the Mitchell concept. Both are available.

Physiology "chooses" which system to use depending on the circumstance it is presented with. Neither is an "accident". The behaviour of complex IV is determined by it's phosphorylation state. Mostly it's phosphorylated and so behaves like the green line from the second graph and ROS are minimised.

If you strip away the phosphates from complex IV it behaves like the red graph and allows a membrane potential of 140-200mV, with associated ROS generation:

"The allosteric ATP inhibition of cytochrome c oxidase is switched on by cAMP-dependent phosphorylation and switched off by [Ca2+]-induced dephosphorylation of the enzyme (Bender and Kadenbach, 2000)."

That's right: Ca2+ ions dephosphorylate complex IV to allow respiration to proceed to a higher membrane voltage with the acceptance of high ROS generation. The gain appears to be the ability to generate more ATP under "stress" situations and this is primarily under hormonal control. Hormonal control is interesting to look at in another post. But for now:

My bottle of calcium borogluconate was stripping phosphates off of complex IV to allow more ATP production in a myocardium poisoned with an inhalation anaesthetic agent, halothane back in the day. The cost would be increased ROS and it's probably a good idea that we stopped doing it.

Peter