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# The virtual laboratory: Oxidative phosphorylation

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### copyright © 1982 - 2006 David A Bender

### Virtual Laboratory main menu

### Click here to run the program

### Click here to see theory pages

In this simulation you can investigate the consumption of oxygen by isolated mitochondria incubated with malate or succinate, with and without an uncoupler, and with and without the addition of a variety of inhibitors and respiratory poisons.

The program will run in a separate window, and at any time you can minimise the program window to check the theory from this page.

As you run the program, you are asked after each simulated experiment whether you wish to save the results to print out. If you save results, they are saved in a file called simout.csv in your temporary file area. The program automatically locates your temporary file area, and displays the path on the opening screen. When you close the program you are given the option of printing out the results you have chosen to save for printing.

### Click the links below to jump to different pages of theory, or just browse down

> ### The mitochondrion
> Measurement of oxygen consumption - the Clark oxygen electrode
> Carriers of the electron transport chain
> The electron transport chain
> The transmembrane proton gradient
> ATP formation
> Respiratory poisons
> Calculating the P:O ratio

The formation of ATP by condensation of ADP and inorganic phosphate in mitochondria is linked to the oxidation of metabolic fuels by the mitochondrial electron transport chain. Under normal conditions this linkage is obligatory; phosphorylation cannot occur without oxidation of substrates and utilisation of oxygen, and conversely the rate of oxidation of substrates, and hence the rate of utilisation of oxygen, is controlled by the availability of ADP to be phosphorylated, and hence by the rate of utilisation of ATP in performing physical and chemical work.

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# The mitochondrion

## Outer membrane

> relatively freely permeable to substrates
> phospholipase
> fatty acid elongation
> triacylglycerol synthesis from fatty acids

## Inter-membrane space

> nucleotide metabolism
> transamination of amino acids
> various kinases

## Inner membrane

> regulation of transport of substrates, etc

## Matrix

> fatty acid oxidation
> citric acid cycle
> variety of other dehydrogenases
> enzymes for mitochondrial replication
> DNA for some mitochondrial proteins

## Cristae

> electron transport
> oxidation of coenzymes linked to reduction of oxygen to water

## Primary particles

> Oxidative phosphorylation (ADP + Pi to form ATP)

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# Measurement of oxygen consumption - the Clark oxygen electrode

The utilisation of oxygen during the reaction is measured using an oxygen electrode, which determines the percentage saturation of oxygen in the reaction mixture. The instrument is calibrated against a buffer saturated with oxygen to set 100% saturation and a buffer totally depleted of oxygen (by reaction with a strong reducing agent such as sodium dithionite) to set 0% saturation.

The reaction chamber is separated from the electrodes by a teflon membrane, which permits oxygen to diffuse from the reaction buffer into the potassium chloride solution that bathes the electrodes: a platinum cathode and a silver anode. A voltage is applied between the electrodes and the resulting current (approx. 1 µA) is proportional to the concentration of oxygen.

At the cathode oxygen is reduced to water:

At the anode metallic silver is oxidised to silver chloride:

After allowing the buffer and substrate to equilibrate, the mitochondrial preparation is added through the injection port, and the consumption of oxygen is measured for a short time, then the ADP and any inhibitors, etc, are added, and oxygen consumption measured until there is no further reaction. The results from a typical experiment are shown here:

In the electrode you will be using in these studies, 100% saturation with oxygen = 1000 nmol O.

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# Carriers of the electron transport chain

Two groups of coenzymes react directly with substrates: the nicotinamide nucleotides and the flavin coenzymes. In addition, the electron transport chain contains ubiquinone, cytochromes and non-haem iron proteins. The nicotinamide nucleotides, flavins and ubiquinone all undergo oxidation / reduction reactions reactions involving transfer of both hydrogen ions and electrons; the cytochromes and non-haem iron proteins undergo oxidation / reduction reactions reactions involving transfer of electrons only.

## The nicotinamide nucleotide coenzymes, NAD and NADP

In NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate), the reactive group is the nicotinamide ring. In the oxidised coenzymes this has a positive charge; reduction is always a two-electron process, resulting in formation of NAD(P)H and an associated proton.

## The flavin coenzymes

In different enzymes flavins may be present as covalently-bound riboflavin, or tightly (but non-covalently) bound riboflavin monophosphate (which is sometimes known as flavin mononucleotide, FMN) or flavin adenine dinucleotide (FAD).

The flavin coenzymes can undergo redox reactions either as a single two-electron reaction or as two single-electron reactions, by way of an intermediate flavin free radical.

## Ubiquinone (Coenzyme Q)

Ubiquinone undergoes a two-electron redox reaction in two single electron steps, via a half-reduced semiquinone radical.

## The cytochromes and non-haem iron proteins

The cytochromes consist of a tetrapyrrole (porphyrin) ring chelating a central iron atom; in the non-haem iron proteins the iron atoms are chelated by sulphydryl groups of cysteine residues and inorganic sulphide (hence the alternative name of iron sulphur proteins). In both cases the iron atom undergoes a single-electron redox reaction.

The haems in different cytochromes differ in the substituents in the porphyrin ring. This affects both their redox potential and also permits different methods of binding to the proteins in different cytochromes:

> Protoporphyrin IX (haem) is non-covalently bound by histidine residues in the protein in haemoglobin, myoglobin, catalase and cytochromes b, b1 and P450.
> Haem C is covalently bound to cysteine residues of the protein in cytochromes c and c1.
> Haem A is anchored in the membrane lipid by a long hydrophobic tail in cytochromes a and a3.

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# The electron transport chain

The carriers of the mitochondrial electron transport chain act to transfer the electrons derived from oxidation of substrates down an electrochemical gradient until finally oxygen is reduced to water. At three stages in the re-oxidation of NADH there is phosphorylation of ADP to yield ATP; in the re-oxidation of reduced flavins there are only two steps at which ADP is phosphorylated to ATP.

A more detailed view of the arrangement of the carriers in the electron transport chain is shown below - each carrier is reduced by the one above it, and in turn is reoxidised by reducing the carrier below, until finally oxygen is reduced to water. This diagram shows the three complexes of carriers involved in the re-oxidation of NADH; each complex is associated with phosphorylation of ADP to ATP. Complex II is not shown here - it is the reaction of reduced flavins with ubiquinone, which is not associated with phosphorylation of ADP to ATP.

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# The transmembrane proton gradient

The carriers of the electron transport chain pump protons across the crista membrane, forming a gradient, with an excess of protons in the crista space, and an excess of hydroxyl ions in the matrix. These protons return to the matrix, down the concentration gradient, through the stalk of the primary particles. This provides the driving force for the ATP synthetase to catalyse the condensation of ADP + phosphate to form ATP.

The creation of the proton gradient is seen most simply in complex I, where there is alternation between coenzymes that carry protons and electrons and those that carry only electrons, so that protons are expelled into the crista space, then acquired from water at the matrix face.

Uncouplers are weak acids that transport the protons back into the matrix directly, bypassing the ATP synthetase - the classic example of an uncoupler is 2,4-dinitrophenol, which is water-soluble when deprotonated, but lipid soluble when protonated, so that it will diffuse across the crista membrane down its concentration gradient. This means that the rate of oxidation of substrates, and utilisation of oxygen, is no longer controlled by the availability of ADP, and in the presence of an uncoupler there is rapid and more or less complete utilisation of oxygen, regardless of the amount of ADP present.

Uncouplers also permit rapid utilisation of oxygen in the presence of compounds that inhibit ATP synthetase, or the transport of ADP into, and ATP out of, the matrix.

Obviously, an uncoupler will have no effect if the electron transport chain itself has been inhibited, since there is no proton gradient to be discharged by the uncoupler.

Click here to view a cartoon of proton transport across the crista membrane with and without an uncoupler.

(You can minimise it to see this screen without closing the program; when you close or end the program this screen will be visible again)

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# The formation of ATP

The formation of ATP by condensation between ADP and inorganic phosphate is a highly endothermic reaction. The ATP synthetase is located in the primary particles on the inner face of the crista membrane; isolated primary particles catalyse hydrolysis of ATP to ADP and phosphate, and you will sometimes see ATP synthetase referred to as an ATPase.

ATP synthetase is a multi-subunit enzyme with three equivalent catalytic sites.

At any given time, each site is at a different stage in the reaction:

> One site is binding ADP and phosphate

> One site is catalysing the condensation of ADP and phosphate, and expelling and water

> One site is expelling ATP, ready to accept ADP and phosphate

As protons enter through the stalk of the primary particle that spans the crista membrane, so they cause a rotation of the central part of the ATP synthetase, forcing each site in turn to proceed to the next step in the reaction.

Electron transport and substrate oxidation are controlled by the availability of ADP

If there is no ADP available to bind to the empty site, then rotation of the central part of the ATP synthetase is not possible, and protons cannot cross the stalk of the primary particle. This leads to an accumulation of protons in the crista space, which, in turn, inhibits any further expulsion of protons by the electron transport chain, so that electron transport (and therefore substrate oxidation) ceases.

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# Respiratory poisons

A wide variety of different compounds act as respiratory poisons, and inhibit the oxidation of metabolic fuels linked to the phosphorylation of ADP to ATP.

> Cyanide and carbon monoxide, well known poisons.
> Sodium azide - another well-known poison
> Antimycin - one of a family of antibiotics produced by Streptomyces spp., some of which are used as fungicides against fungi that are parasitic on rice.
> Amytal (amobarbital) - a barbituric acid derivative widely used as a sedative or hypnotic drug.
> Rotenone - the main insecticidal compound in derris powder, extracted from the root of the leguminous plant Lonchocarpus nicou.
> Oligomycin - one of a family of antibiotics produced by Streptomyces spp., which is of little or no therapeutic use.
> Atractyloside - a toxic alkaloid from the rhizomes of the Mediterranean thistle Atractylis gummiferra. It competes with ADP for binding to the adenine nucleotide transporter.
> Bongkrekic acid - a toxic antibiotic formed by Pseudomonas cocovenans growing on coconut. It anchors the adenine nucleotide transporter at the inner face of the membrane, so that ATP cannot be transported out, nor ADP in.

You will be able to perform experiments with several of these compounds, and you should be able to deduce how they act from your results.

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... and a respiratory stimulant

2,4-dinitrophenol is a weak acid; the ionised form is water-soluble, whereas the protonated (unionised) form is lipid-soluble and will diffuse through membranes. It can thus act to transport protons from one side of the mitochondrial membrane to the other, as can a variety of weak acids.

Consumption of dinitrophenol leads to an increased metabolic rate, high oxygen consumption and a high body temperature. As you perform experiments with dinitrophenol added to your mitochondrial preparation you should be able to deduce how its acts to stimulate substrate oxidation.

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# Calculating the P:O ratio

For normal incubations (i.e. when there is no inhibitor or uncoupler added) you should calculate the ratio of ADP phosphorylated : atoms of oxygen consumed; this differs for different substrates, and you should work out why this is so.

If you save results to print out when you finish running the program, you will receive a print out like that shown below for each saved experiment.

You should draw straight lines, as shown above, to determine the % oxygen saturation:
A) at the time that the mitochondrial preparation is added
B) at the time that ADP is exhausted

Then measure the distance from 0 - 100% oxygen saturation

In this example, by measurement (on the original printed graph):
100% oxygen saturation (1000 nmol O) = 78 mm

point A = 72.5 mm = 72.5 / 78 = 0.929 x 1327 nmol O = 1233 nmol O

point B = 40 mm = 40 / 78 = 0.512 x 1327 nmol O = 680 nmol O

Hence, difference = 1233 - 680 = 552 nmol O

You should be able to calculate the P:O ratio from this value (the amount of oxygen used) and the amount of ADP you added in the experiment.

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