If there's one thing that mitochondria thrive on, its oxygen. All of it is consumed by cytochrome oxidase, the last enzyme in the electron transport chain which drives ATP production. If cells relied on diffusion alone to supply them with their oxygen needs, then there would not be enough to keep up with demand. So oxygen carrying molecules, such as haemoglobin and myoglobin,evolved to transport oxygen to where it is needed. However as Jonathan and Beatrice Wittenberg explain, researchers know very little about the conditions necessary for oxygen to reach cytochrome oxidase(p. 2082).
As oxygen travels through the body it exerts a pressure in the mixture of gases in the lungs, or in solution, known as the partial pressure. Oxygen bound to haemoglobin in the blood diffuses down a steep pressure gradient into tissues as blood travels through capillaries. Next oxygen diffuses into the mitochondria. By reducing the oxygen pressure to levels below which mitochondria would not get enough oxygen without the help of haemoglobins, the Wittenbergs hoped to find the oxygen partial pressure necessary for oxygen uptake by mitochondria from hard working pigeon hearts. Also, would myoglobin in the heart muscle need to bind to mitochondria to deliver oxygen? To extract mitochondria for their study, the team delicately ground up the heart muscle tissue with a homogeniser and dissolved away the toughest tissue with enzymes;then, they released the mitochondria from the cell fragments and put them in a nourishing solution.
To show that myoglobin doesn't need to bind to the surface of mitochondria to deliver its oxygen, they used six different haemoglobins in the solution to deliver the oxygen: one each from horse, an insect, and soy bean, and three from molluscs. Each binds and releases oxygen at very different rates. Using a method called spectrophotometry, where a light is shone through biological samples and the light absorbed at each wavelength is measured, the team could tell how oxygenated the haemoglobins were since they absorb different light wavelengths depending on how much oxygen they are carrying. Despite differences in the speed with which oxygen bound to and was released from the haemoglobins, the mitochondria still took up oxygen at the same rate, showing that the haemoglobins didn't bind to the surface to deliver their cargo.
To find what oxygen partial pressure kept cytochrome oxidase functioning normally, they measured the saturation of each of the haemoglobins with oxygen and how it decreased as the mitochondria used oxygen up. From this they calculated oxygen pressure, which is directly related to haemoglobin saturation. When oxygen uptake was half its maximal rate, they found that the oxygen pressure at the surface of the mitochondria was very similar for all the haemoglobins, around 0.0053 kPa, despite their different reaction kinetics. This is much smaller than the pressure measured previously in working hearts, around 0.32 kPa. This means that even when a heart muscle is working flat out, such as during flight, the mitochondria will still have plenty of oxygen available to generate ATP.
Because oxygen uptake also levelled out as they increased the concentrations of the haemoglobins, the team suspect that there is just enough myoglobin present to support the cell, but not more, indicating that cells optimise oxygen delivery. `The results were not unexpected', Jonathan Wittenberg explains. Despite this, he says, `there is still a lot we don't understand about oxygen transport in heart and muscle'.