Oxidative phosphorylation: regulation and role in cellular and tissue metabolism

Abstract

Oxidative phosphorylation provides most of the ATP that higher animals and plants use to support life and is responsible for setting and maintaining metabolic homeostasis. The pathway incorporates three consecutive near equilibrium steps for moving reducing equivalents between the intramitochondrial [NAD⁺ ]/[NADH] pool to molecular oxygen, with irreversible reduction of oxygen to bound peroxide at cytochrome c oxidase determining the net flux. Net flux (oxygen consumption rate) is determined by demand for ATP, with feedback by the energy state ([ATP]/[ADP][P_i ]) regulating the pathway. This feedback affects the reversible steps equally and independently, resulting in the rate being coupled to ([ATP]/[ADP][P_i ])³ . With increasing energy state, oxygen consumption decreases rapidly until a threshold is reached, above which there is little further decrease. In most cells, [ATP] and [P_i ] are much higher than [ADP] and change in [ADP] is primarily responsible for the change in energy state. As a result, the rate of ATP synthesis, plotted against [ADP], remains low until [ADP] reaches about 30 μm and then increases rapidly with further increase in [ADP]. The dependencies on energy state and [ADP] near the threshold can be fitted by the Hill equation with a Hill coefficients of about -2.6 and 4.2, respectively. The homeostatic set point for metabolism is determined by the threshold, which can be modulated by the PO2 and intramitochondrial [NAD⁺ ]/[NADH]. The ability of oxidative phosphorylation to precisely set and maintain metabolic homeostasis is consistent with it being permissive of, and essential to, development of higher plants and animals.

Keywords: ATP synthesis; energy metabolism; exercise; metabolic homeostasis; oxidative phosphorylation.

Figure 1. A skeleton representation of glycolysis

Each reaction in the pathway from glucose (Glu) to lactate is represented with an arrow indicating whether it is fully reversible and near equilibrium (↔) or irreversible (→). There are three irreversible steps responsible for regulating the pathway, hexokinase (HK) or glucokinase (GK), phosphofructokinase (PFK), and pyruvate kinase (PK). All of the other reactions are freely reversible (free energy change near zero) and operate either in the forward (glycolysis) or reverse (gluconeogenesis) direction as needed. Each of the irreversible reactions is accompanied by an energy loss (−free energy change) of more than 12.5 kJ mol¹ as heat. The irreversible steps determine the net forward flux through that part of the pathway and act as valves controlling the direction of the flux, i.e. PFK determines whether carbon from glucose is stored as glycogen or metabolized to pyruvate for oxidation by the citric acid cycle (CAC) or exported as lactate.

Figure 2. A schematic diagram of oxidative phosphorylation

Most of the reducing equivalents used in oxidative phosphorylation come from intramitochondrial NADH, which is produced by the citric acid cycle, fatty acid oxidation, and amino acid metabolism. The intramitochondrial [NAD⁺]/[NADH] ratio is regulated and is typically maintained at a redox potential near −0.35 ± 0.03. All of the reactions within oxidative phosphorylation are near equilibrium except the reduction of molecular oxygen to the bound peroxide in cytochrome c oxidase. The redox components of the respiratory chain are organized in groups with half‐reduction potentials near −0.3 V, −0.00 V, 0.25 V, and 0.6 V, the most positive being the bound peroxide intermediate of the oxidase. Within each group of redox components the exchange rates sufficiently exceed the net flux through oxidative phosphorylation that they form an isopotential ‘pool’ of reducing equivalents. Feedback by the energy state is applied equally and independently to each step, resulting in 3 stage amplification of the signal.

Figure 3. The dependence of the net flux through oxidative phosphorylation on [ADP] when [NAD⁺]/[NADH] and [O₂] are constant, as predicted by the model

A, as [ADP] increases the net flux, expressed as the turnover number (TN) of cytochrome c, remains very low until the [ADP] is 30–40 μm and then the rate begins to increase very rapidly with further increase in [ADP]. This pattern is typical of functions (y‐axis) which have an exponential dependence on a parameter (x‐axis). In biology this is most commonly discussed in the binding of oxygen to haemoglobin (Hill equation) and other cooperative reactions while in electronics it often discussed in relation to control circuits (Zener diodes, for example). B, the threshold region of the curve and its fit to the Hill equation. There is clearly an excellent fit of the date (χ² = 0.006) when the fitting parameters are V _m = 16.5, k = 79, and the Hill coefficient (n) = 4. This indicates for these conditions the net flux increases as the fourth power of the ADP concentration ([ADP]⁴). It must be noted that although for these conditions the behaviour gives a good fit to the Hill equation, and the flux is changing as [ADP]⁴, the underlying mechanism is different from cooperative binding as observed in oxygen binding to haemoglobin.

Figure 4. The dependence of the net flux on energy state (A) and [ADP] (B) at constant [O₂] but different [NAD⁺]/[NADH] levels

A, the net flux, expressed as the turnover number (TN) for cytochrome c, is plotted against the energy state. The [ATP] and [ADP] are constant at 6 mm and 3 mm, respectively, while the predicted behaviour is was calculated for [NAD⁺]/[NADH] values of 0.3, 0.1, and 0.03. At each [NAD⁺]/[NADH] value, as the energy state increases the rate of ATP synthesis (net flux) initially falls rapidly but then slows progressively and asymptotically approaches zero at high energy states. Decrease in the [NAD⁺]/[NADH] (increased reduction of the NAD couple and more negative redox potential) shifts the curves to higher energy states by a factor of 2.1 for each 10‐fold decrease in the [NAD⁺]/[NADH]. B, the cytochrome c turnover (net flux) is plotted against [ADP] for cells that do not have significant content of creatine phosphate or arginine phosphate. In these cells the [ATP] and [P_i] are a few millimolar whereas the [ADP] is tens of micromolar , and most of the change in energy state is through change in [ADP]. As seen also in Fig. 3 A and B, the turnover number for cytochrome c remains very low until a threshold value near 30 μm is reached, after which the flux increases very rapidly with further increase in [ADP]. Decrease in [NAD⁺]/[NADH] (reduction of the NAD pool) lowers the threshold [ADP] and increases the steepness of the rise in cytochrome c turnover number with increase in [ADP] above threshold.

Figure 5. The effect of inclusion of arginine kinase or creatine kinase on the regulation of oxidative phosphorylation when [O₂] and [NAD⁺]/[NADH] are held constant

The cytochrome c turnover number (TN) is plotted against [ADP] for cells without either arginine or creatine kinase (no Art or Crt), with arginine kinase and 40 mm total arginine concentration (Art = 40 mm) or with creatine kinase and 40 mm total creatine (Crt = 40 mm). In all cases the [O₂] was held constant at 60 μm and the [NAD⁺]/[NADH] constant at 0.1. Both the arginine and creatine systems result in a dramatic increase in the flux attained for each [ADP] but have little effect on the threshold. The latter is because the [P_i] near the threshold (resting conditions) is very similar for most cells. The contributions of ArP or CrP hydrolysis to [P_i] provides a more effective response (higher rates at similar [ADP]) to metabolic challenges such as increased work rate. Note that if the cytochrome c turnover were plotted against energy state there would be no effect of adding these enzymes since their contribution is entirely due to the change in [P_i] that accompanies the hydrolysis of ArP or CrP.

Figure 6. The dependence of oxidative phosphorylation on [O₂] in the microenvironment

The predicted behaviour of the energy state has been calculated for a constant reduction of the NAD couple ([NAD⁺]/[NADH] = 0.1) and constant cytochrome c turnover number (TN; 6 s⁻¹) while [O₂] was decreased from 80 μm to zero. The resulting changes in energy state, [ADP] and [AMP] are plotted as a function of [O₂]. All three parameters increase continuously as the [O₂] decreases, with the largest increase occurring in [AMP]. Under physiological conditions the mean intracellular oxygen concentrations are typically 50–60 μm (13, 60) and if this decreases to 10 μm, the tissue is seriously hypoxic.

Figure A1. A schematic diagram of the kinetically important reactions involved in the reduction of oxygen by cytochrome c oxidase

The individual reaction intermediates are designated by the Roman Numbers I through V and these numerals are used for setting up the differential equations describing the rates of formation and removal of these intermediates. The steady state rate expression is then derived assuming the rates are equal and there is no change in the concentrations of the intermediates. Only the reactions of oxidative phosphorylation from cytochrome c to oxygen are presented. All of the electron transfer reactions are assumed to occur across the same difference in redox potential (energy coupled) for which ΔG = −46.183 kcal V⁻¹ (193.23 kJ V⁻¹).