1. Predictions regarding respiratory control and p/o ratio.

Assume that: As 2e pass through the respiratory chain, 4H+ are ejected from the mitochondrial matrix at complex I, 4H+ at complex III, and 2H+ at complex IV, for a total of 10 H+ ejected per 2 e transferred from NADH to ЅO2. Four H+ enter the matrix for each ~bond of ATP synthesized.

Based on these assumptions, what would be the predicted P/O ratio, i.e., the number of  ATP molecules synthesized per 2 e^ transferred from NADH to O₂? _________  

Complex II of the respiratory chain (Succinate Dehydrogenase) catalyzes transfer of 2 e^ from succinate to coenzyme Q (via FAD and Fe-S centers). What would be the predicted P/O ratio with succinate as electron source instead of NADH? _________     

2. Atp production with aerobic metabolism of glucose

For this calculation, assume that oxidative phosphorylation in mitochondria yields 2.5 ~P bonds of ATP per NADH oxidized (except 1.5 ~P for the NADH from Glycolysis), or 1.5 ~P bonds of ATP per coenzyme QH₂ oxidized. Fill in the number of NADH, QH₂, and ~P bonds of ATP produced per glucose at each stage of the process, and add up the total.  Question: How does the yield of ~P bonds in aerobic metabolism compare to the yield of ~P bonds from catabolism of glucose in an anaerobic organism?

	Per glucose molecule metabolized
Pathway	NADH produced	QH2 produced (via FADH2)	~P bonds ATP or GTP direct	~P bonds 1.5 or 2.5 per NADH in oxphos	~P bonds  1.5 per QH2 in oxphos	Total ~P bonds
Glycolysis Pathway
Pyruvate Dehydrogenase
Krebs Cycle
Sum of Pathways

3. Determination of respiratory control and p/o ratios

An oxygen electrode recording generated by an RPI undergraduate student is provided below. In this experiment, isolated rat liver mitochondria were suspended in buffered medium containing 200 mM sucrose (for osmotic support), 7.5 mM inorganic phosphate, and 7.5 mM succinate (for input of electrons to the respiratory chain via Succinate Dehydrogenase). When indicated, 0.6 or 1.5 mole ADP was added. Assume that the 2.2 ml total volume was equilibrated with air at the outset, so the initial [O₂] was 0.24 mM. P/O ratios are calculated per oxygen atom (or per 2 electrons transferred), so the total oxygen present from the beginning of the recording is 2.2 ml X 0.48 mol/ml of Ѕ O₂, or 1.06 mol O. For scaling, you need to estimate how many divisions (or mm) across the chart paper corresponds to this amount of O.

What is the respiratory control ratio, based on the ratio of slopes b/a during phosphorylation of ADP (b), and before addition of ADP (a). Respiratory control ratio: _____________

Some questions to be answered:

• Why is the respiration rate very low in the absence of ADP? What might be the basis for the finding that the rate of oxygen consumption is not zero in the absence of ADP?

• How might you explain the gradual change of slope (non-linearity) when ADP is nearly exhausted?

• How could you estimate the K_M for oxygen of cytochrome oxidase (complex IV) from the recording given?

Calculate the P/O ratio from the amount of ADP added, divided by the amount of oxygen used up while phosphorylating the ADP to ATP. Since the rate of oxygen consumption slows as [ADP] becomes low, you must use a ruler to estimate the intersection of the slopes before and after exhaustion of ADP. P/O ratio estimated with succinate as e^ source (molecules of ATP synthesized per 2e^ transferred from succinate to ЅO₂): _____________