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Lecture Comments (20)

2 answers

Last reply by: Sally Reina
Tue Sep 5, 2017 1:51 AM

Post by Sally Reina on August 29 at 01:04:45 AM

Hi Professor Raffi Hovasapian,

For Complex IV, it pumps out 2H+, but I am confused by the figure because it showing 4H+ being pumped into the intermembrane space. Can you explain this please?


1 answer

Last reply by: Professor Hovasapian
Fri Apr 7, 2017 6:52 PM

Post by Nikhat Siddiqi on March 25 at 02:42:34 PM

Can you please give me a lecture on "Transport across biomembranes: free energy content of transmembrane concentration gradients for uncharged and charged solutes mathematical relationship"


0 answers

Post by Phil Beauchamp on July 11, 2014

What about charge balance?  2 electrons total -2 in charge move from a NADH through the electron transport chain but they are moving +10 to the IM?  Also, when the electrons move, they seem to usually move as hydride, moving a proton with them (or picking up one after a single electron transfer) yet the hydrogen seems like it is considered as H+.  It is a little confusing.

0 answers

Post by Professor Hovasapian on September 10, 2013

Hi Vinit,

The answer is  thermodynamics.  The oxidation of 1 mol of NADH to NAD+ has 220 kJoules of Free energy. Some of this energy is lost simply by the inherent inefficiency of the proton pump. However, most of it ( about 200 joules is used to pump out those protons. This 200 kjoules is used to pump 10 moles of protons. So 1 molecule of NADH oxidized allows 10 H+ protons.

So the number 10 just happens to reflect the Total Free Energy available to do work, and the fraction of that which actually does the work of pumping out protons.

Hope that helps.


1 answer

Last reply by: Professor Hovasapian
Tue Sep 10, 2013 8:18 PM

Post by Vinit Shanbhag on September 9, 2013

Thanks for the answer. Another question I had was, why only 10 electrons are pumped out per NADH molecule, I know that 4 protons make 1ATP by the ATP synthase, so 10 will make 2.5. Thanks.

0 answers

Post by Vinit Shanbhag on September 7, 2013

Hi, forgot to mention that your lectures r really helpful. Thanks a lot.

1 answer

Last reply by: Professor Hovasapian
Sun Sep 8, 2013 4:26 AM

Post by Vinit Shanbhag on September 7, 2013

difference between atp synthase and atpase?
does krebs cycle require oxygen? how is it dependent on ETC for its byproducts?

1 answer

Last reply by: Professor Hovasapian
Fri Apr 26, 2013 1:44 AM

Post by Kenshin Kenshin on April 25, 2013

Hi, I was wondering in the equilibrium section of the new pchem vids you were going to make are you going to go over the acid/base equilibrium rxn in the more in depth version for pchem compared to gen chem?

5 answers

Last reply by: Professor Hovasapian
Sun Apr 21, 2013 10:04 PM

Post by Brian Phung on April 21, 2013

Hi I was wondering if there would be any vid on signaling pathway?

Oxidative Phosphorylation II

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Complex III 0:19
    • Complex III Overview
    • Complex III: Step 1
    • Complex III: Step 2
  • Complex IV 8:42
    • Complex IV: Cytochrome Oxidase
  • Oxidative Phosphorylation, cont'd 17:18
    • Oxidative Phosphorylation: Summary
    • Equation 1
    • How Exergonic is the Reaction?
    • Potential Energy Represented by Transported H⁺
    • Free Energy Change for the Production of an Electrochemical Gradient Via an Ion Pump
    • Free Energy Change in Active Mitochondria

Transcription: Oxidative Phosphorylation II

Hello and welcome back to, and welcome back to Biochemistry.0000

Today, we are going to continue our discussion of oxidative phosphorylation.0004

In the previous lesson, we looked at complex 1, complex 2; we gave a little bit of an overview of what was going on.0008

Now, we are going to move on to the rest of the electron transport chain beginning with complex 3.0014

OK, complex 3, let's see what we have here.0020

Now, we have all of these high-energy electrons that are collected by the molecule ubiquinone, and now, it is in the form of ubiquinol, its reduced form.0033

Now, it is going to pass on its electrons.0042

It is going to pass its electrons to cytochrome c, and here is how it is going to be.0048

Let's go ahead and give a name to this.0055

This is called cytochrome bc1 complex.0059

It is also called its formal name Q:citochrome c oxidoreductase.0069

This is the name of the complex; normally, we just call it the cytochrome bc1 complex.0087

That is the common name; the formal name, when you see this :Q to cytochrome, that means something is being transferred from Q to cytochrome c, and it is an oxidoreductase.0092

Oxidoreductase, you remember from the classification, it involves the transfer of electrons.0103

OK, in the complex 3, in this particular case, electron transfer, it takes place in 2 steps.0110

It is really an unusual complex.0122

OK, the first step, what you have is a molecule of QH2.0130

Now, it has its electrons; what it does, it releases its hydrogen ions into the inter-membrane space.0136

Again, this is part of the proton pump quality of complex 3.0145

It releases the 2 Hs into here.0150

Well, one of its electrons, it gives it over to ultimately cytochrome c, and we will talk about the actual sequence in a second; but it goes from Q to cytochrome c.0155

The other electron, now, what it does is it gives it over to another molecule of just plain old Q, and what ends up happening is this Q ends up turning into the QH radical that we talked about earlier.0166

Now, another molecule of QH2 diffuses through the bilayer.0182

It release its 2 hydrogen ions into solution.0187

Now, we have a total of 4 hydrogen ions being passed into the inter-membrane space.0192

It, again, gives 1 electron to Q.0198

It passes it to cytochrome c; it gives its other electron over to this radical, and this radical, now, ends up becoming QH2.0204

In the second step, 1 molecule of QH2 is used up.0212

1 molecule of QH2 is created; there is no net gain or loss of QH2.0218

Ultimately, it is only this 1 molecule of QH2 that ends up passing its electrons- 1 here to cytochrome c, the other here, another 1 to cytochrome c, the other here.0225

We have actually passed 2 electrons, and we have pumped 4 protons into the matrix.0235

That is what is happening here; complex 3, well, it is definitely not altogether that easy to follow.0242

There is a lot going on with complex 3; let's talk about what happens here.0249

Electron transfer takes place in 2 steps.0253

The first step, a molecule of QH2 - the reduced form of ubiquinone, which is called ubiquinol - releases 2 hydrogen ions to the inter-membrane space.0257

OK, that is the P side- the positive side.0279

Now, 1 electron passes as follows.0284

This is the inner workings of the complex: QH2 to an iron sulfur protein to cytochrome c1 and ultimately to cytochrome c.0292

This is the actual path of the electron, and 1 electron passes as follows: QH2 to cytochrome b to Q, which, now, becomes QH radical.0307

OK, the first electron passes from QH2; this QH2 and this QH2, they are the same.0347

One electron, it goes from an iron sulfur protein to cytochrome c1 to cytochrome c.0354

It is, now, here; the other electron passes to a cytochrome b and then, to Q, which, now, becomes QH- this Q radical.0360

That is what that becomes; now, for step 2, another QH2 molecule, it does the same thing.0371

It does the exact same thing; it does the same thing.0390

It releases 2 hydrogen ions into the inter-membrane space.0400

Well, 1 of the electrons passes as QH2, iron sulfur protein, cytochrome c1 to cytochrome c, and the second electron, it passes as QH2 - this is the same QH2, 1 electron goes 1 way, 1 electron goes the other way - to cytochrome b; but now, it goes to the radical, which, now, becomes our QH2 again.0410

In step 2, 1 QH2 is used, and 1 is created.0468

So, there is no net gain or loss.0490

OK, and like complex 1, complex 3 is a proton pump, hydrogen ion pump- that is it.0494

That is all that is going on there; OK, now, let's take a look and move on to complex no. 4.0520

OK, this is complex 4.0529

The movement is now from cytochrome c.0537

The electrons are going to finally make their way to O2- the final destination of these electrons.0541

The name of this is cytochrome oxidase.0547

OK, let's talk a little bit about...I want to say something about the oxidase.0556

An oxidase is a general class of enzymes that catalyze oxidations, where O2 is the electron acceptor, but O, itself, oxygen atoms, do not appear in the product.0565

Both atoms of oxygen end up being reduced to water.0622

One atom takes 2 electrons and 2 hydrogens to become 1 molecule of water.0626

The other oxygen atom takes another 2 electrons and another 2 hydrogen ions to become water.0630

Oxygen, itself, does not show up anywhere; it is pure oxidation up here in the product.0637

OK, now, let's see what actually goes on here.0645

In this particular case, this is also going to, sort of, happen in 2 steps.0654

What you see here, though, is the final result.0659

Now, you have this cytochrome c; all of the electrons have been passed to cytochrome c.0665

Cytochrome c is like a ubiquinone in a sense that it is a mobile electron carrier.0670

Again, complex 3, complex 4, we need to get the electrons from here to here.0678

Cytochrome c is what moves on the lipid bilayer, through the lipid bilayer.0682

And again, it is just another molecule that carries electrons.0690

It carries/has 2 electrons, moves over here, gives up 2 electrons, moves back, takes 2 electrons, just back and forth- that is it.0694

It does the same thing; it just an electron shuttle is what it is.0700

The net effect is the following; 4 cytochrome cs pass their electrons to, this time, a copper-copper center, passes it on to a heme group, passes the electron to another heme group, passes the electron actually to a heme copper center, and now, these electrons, here is where O2 comes in.0705

O2 + 4 hydrogen ions plus these 4 electrons is reduced to 2 molecules of water.0730

In the process, also 4 hydrogen ions is pumped from the matrix into the inter-membrane space.0737

This is the net effect of what complex 4 does.0744

Cytochrome c passes its electrons ultimately to O2, reduces both atoms of oxygen in the O2 molecule to water.0748

OK, that is very, very important- 4 protons, 4 electrons, 2 each to form waters.0758

Now, we will go ahead and do a breakdown of the individual process.0766

Again, an oxidase is a general class of enzymes that catalyze oxidations where O2 is the electron acceptor, but O atoms do not appear in the product.0770

OK, now, this is different from the mixed-function oxidase that we saw earlier for the phenylalanine hydroxylase reaction.0779

Remember the first reaction of the catabolism for phenylalanine?0788

That enzyme, the phenylalanine hydroxylase, is also called a monooxygenase because it uses O2 as the electron acceptor, but one of the oxygens actually ends up in the product.0794

The phenylalanine becomes tyrosine; the other oxygen atom ends up being reduced to water.0808

In this case, an oxidase, both atoms end up getting reduced to water.0814

That is the difference; even though we call this an oxidase, that other remember, we called it a mixed-function oxidase?0820

That is a really unfortunate, sort of, common name for it- mixed-function oxidase.0828

You will often hear it called a mixed-function oxidase.0831

Really, it is more proper to refer to it as a monooxygenase.0837

This is strictly an oxidase; I know, enzyme names, it is enough to make you want to pull your hair out.0841

Sorry about that; OK, now, let's go ahead and talk about this.0847

Let's see; the first step, we have 2 cytochrome c molecules.0857

Two electrons are passed to this copper-copper center, and then, these electrons are passed to something called heme A.0858

These electrons are passed to something called heme a3.0867

Actually, I am going to draw it a little bit this way.0870

I am going to go to heme a3CuB.0874

It is a heme copper center, and from here, the electrons are passed to O2.0903

However, the O2, it actually becomes O22-.0912

It is not fully reduced yet; now, 2 more cytochrome cs deliver 2 more electrons, and with 4 hydrogen ions taken up from solution, taken up from the matrix, this O22- now, it becomes 2 molecules of water.0920

OK, the 2 electrons that are passed from the 2 cytochrome cs only produces O22-.0968

Two other electrons from 2 other cytochrome cs pass through the same process, and now, the O22- is converted to 2 molecules of water.0974

And, of course, along the way - again, complex 4 is a proton pump - 2 H+s are transported into the inter-membrane space.0986

We had 4 protons from complex 1 being transported, 4 protons from complex 3 being transported, and now, 2 protons from complex 4 being transported.1010

This is all from the transfer of 2 electrons from NADH from the beginning of the electron transport chain.1019

For every 2 electrons that make it through the electron transport chain, 10 hydrogen ions are transported across the membrane.1025

That is a lot; that is a lot.1033

OK, now, let's see what it is that we have got here.1037

We have done complex 1, complex 2; now, let's go ahead and do a quick little summary, so that we know where it is that we stand.1042

NADH goes ahead and delivers its electrons to complex 1, and these electrons are passed to ubiquinone.1051

Four protons are pumped into the inter-membrane space.1059

Let's do this in...that is OK; let's go ahead and do this in black.1065

We have 4 hydrogen ions there; the succinate passes its electrons through FAD, again, to ubiquinone.1070

Complex 2 is not a proton pump; complex 3, the ubiquinone passes its electrons through complex 3 to cytochrome c, and in the process, we have pumped in, again, 4 hydrogen ions from the matrix to the inter-membrane space.1080

Cytochrome c passes its electrons through complex 4.1098

The electron transport chain, it reduces a molecule of O2 to 2 molecules of water.1102

Here, they show the stoichiometry is 1/2, but that is not a problem; and in the process, we end up transferring 2 hydrogen ions.1109

Now, we have 4, 4, 2; we have 10 hydrogen ions in the inter-membrane space.1119

This is the positive side; this is the negative side.1124

These protons, they want very badly to go over here both for concentration reasons and for potential reasons.1128

There is a build-up of charge, and there is a concentration gradient.1134

These protons fly through this molecule called ATP synthase.1138

Downhill, that extra energy is used to actually produce the ATP from adenosine diphosphate and inorganic phosphate.1143

Let's take a look exactly what is going on here, so equation 1.1154

Let's see if we can quantify this a little bit.1159

We have NADH + H+ + 1/2 O2 goes to NAD+ + H2O.1163

There is our H2O; there is our H2O.1175

This is per mole of NADH; per mole of NADH, we produce 1mol of water.1179

Perhaps it is better written if you like, if you do not like this 1/2, it is up to you because it might be better written as 2 NADH.1186

Each NADH deliver 2 electrons; 2 NADH, we have delivered 4 electrons.1202

We have pumped 20 hydrogen ions; we are going to end up producing this many waters.1207

2 NADH + 2 H+ + O2 produces 2 NAD+ + 2 H2O.1212

OK, I guess the question is: "how exergonic is this reaction?".1227

This flow of electrons, again, we said that it is exergonic.1237

It is this giving up of energy, that energy is what is used to actually produce the proton gradient that ends up ultimately falling back down.1240

That energy is used to fall back down; that is where that energy is used to produce the ATP.1252

Well, we want to see how exergonic this reaction is, the transfer of electrons.1258

How exergonic is this reaction?1264

Well, we know that ΔG is equal to -N x F x that, right?1272

The free energy of a particular process when there is a potential difference is the number of electrons transferred times the Faraday constant times the actual potential difference.1283

Well, let's see what this is; let's do a little bit of a calculation here.1296

Let's rewrite the equation: ΔG is equals to -N x F x that.1300

Well, we know what N is; we are talking about the transfer of 2 electrons.1310

We know what the Faraday constant is; it is 96,485J/V.mol.1315

OK, now, the...that is OK.1326

I will do it here; the potential for the NAD+ NADH, oxidation-reduction pair is -0.320V, and the potential for the O2 to H2O pair, that equals 0.816V.1332

The net, the reaction, this minus that, this minus that, this minus the minus that, it is 0.816 + the 0.320 gives me a total of 1.14V.1359

That is the potential difference here and the transfer of these electrons across the electron transport chain.1374

1.14V- that is huge, absolutely huge.1380

Now, let's go ahead and put this together; our ΔG, the amount of free energy that we are actually releasing that is available for other work is going to be -2 x 96,485J/V.mol x 1.14V.1384

And now, what I am going to do is I am going to multiply this; because this is per mole, we wrote our equation as 2 NADH.1411

I am just going to go ahead and multiply this by 2 because I want to do it for 2 moles.1418

When you end up doing this, you end up getting -440kJ.1425

2mol of NADH ends up producing, ends up releasing 440kJ of free energy.1435

This free energy is what is used to transfer the protons from the matrix to the inter-membrane space.1446

Now, this free energy is what is used to pump hydrogen ion out of the matrix.1454

Again, this 440kJ, this is what is coming from just those 4 moles of electrons traveling through the electron transport chain, from complex 1 all the way to complex 4.1480

In the process, in reaching oxygen, they actually released 440kJ of energy that can be used for something else.1498

This free energy is what is used to pump the hydrogen ions out of the matrix and into the inter-membrane space against both a concentration and an electrical gradient.1508

In other words, you have this membrane, and here, you have the matrix; and here, you have the inter-membrane space.1540

Well, you are trying to separate, you are trying to take all of this hydrogen ion and bring them all to this side.1553

You are trying to create; you are doing something that is unnatural.1562

It is not going to happen spontaneously; therefore, you need energy to actually do this.1566

You need to put in energy to run a process that is not going to happen naturally.1570

Naturally, the system wants to be at equilibrium; what you are doing is you are taking all these hydrogen ions, and you are pumping them, and you are putting them on 1 side of a barrier.1577

Now, you have a whole bunch of positive charge, a whole bunch of negative charge.1585

Not only have you separated charge, but you have also separated species.1589

Now, you have hydrogen ion here, no hydrogen ion here.1593

There is a concentration gradient, and there is an electrical gradient; you need energy to support that to make sure that it all stays on this side.1597

You are creating a bunch of potential energy, and now, when we open the floodgate of ATP synthase, all of that potential energy is going to release and create ATP.1605

All of this energy is used to do this, to run an unnatural process, to create this gradient, and as you create the gradient, you are making it actually harder and harder to pump protons.1614

As you separate the charge, as you move the concentration of hydrogen ion from 1 side to the next, you are making it harder for more of it to happen.1626

That is where this energy is going; OK, now, let's see what we have got.1635

Let me see; OK, now, the potential energy represented by these transported hydrogen ions, which are, now, ready to fall back down their gradient, is 2-fold.1645

You have the chemical part; that is the concentration difference.1698

That is the concentration of hydrogen ion on 1 side, virtually no concentration of hydrogen ion on the other side, and it is also electrical.1706

It is a separation of charge; you are creating a positive charge on one side of the membrane and negative charge on the other.1718

This is a lot of potential energy here; OK, now, we have an equation.1725

The free energy change for the production of an electrochemical gradient like this when you actually do this, when you pump an ion from one side of a membrane to the other side of the membrane, we have an expression for actually the free energy that is available from this.1736

The free energy change for the production of an electrochemical gradient via an ion pump is the following.1760

ΔG is equal to RT times the natural logarithm of c1 / c2 + Z, faraday constant, delta phi.1785

Let's talk about what some of this is; now, c1 and c2, they represent the concentrations of the ions on either side of the membrane.1798

c2 represent the concentrations of the hydrogen ion in the 2 compartments, and c1 is greater than c2.1809

In other words, we need this thing to be greater than 1.1835

c1, the concentration 1, that is the concentration of hydrogen ion in the inter-membrane space.1838

Concentration 2 is the concentration of hydrogen ion in the mitochondrial matrix.1844

OK, Z is the absolute value of the charge of the ion, which in this case is just +1.1851

It is just +1 in this case.1873

Hydrogen ion...well, yes, delta phi, now, delta phi is the transmembrane potential difference in volts.1877

In other words, once we actually have that separation of charge, a whole bunch of hydrogen ion, a whole bunch of positive, a whole bunch of negative, there is a potential difference.1900

We can measure that potential difference, and in this particular case, we actually have all of these numbers.1907

In active mitochondria, as it turns out - well, we are not going to worry about what the potential difference is, let's just move on to... - once we actually put the numbers in, this ΔG is approximately equal to about 20kJ/mol of hydrogen ion.1915

Well, since 2 electrons transfer 10 hydrogen ions, they pump - OK - 10 x 20.1950

This is about 200kJ/mol of NADH.1967

Well, since we decided to use the stoichiometry of 2 NADH, this is about 400kJ.1974

With 2mol of NADH, in other words, with 4 electrons transferred, with 20 hydrogen ions pumped across the membrane, ΔG approximately equals about 400kJ.1984

We said that the electrons, in moving through the electron transport chain, they release about 440kJ of energy per 2mol of NADH.2008

We have taken that energy, and we have preserved it.2024

Instead of just letting it dissipate as heat, we have used about 400kJ of that energy to actually produce this proton gradient.2028

Now that that proton gradient this is potential energy, there is this 400kJ of energy that once these protons, once we open up the ATPase of molecule, these protons are going to want to fall back down their gradient.2038

They want to get to the other side from the inner membrane space.2053

Here is the membrane; they want to get to the matrix.2057

There is all of this 400kJ of energy that we have used from the 440 to pump to protons, but now that they are there, we have created this gradient.2061

Now, they want to fall back down; now, this 400kJ is actually potential energy, which can be converted into work.2070

This work is what produces the ATP; it is what builds the ATP from the ADP and the inorganic phosphate.2078

Of the 440kJ from the electron transport chain, about 400kJ is preserved as the proton gradient, and this is ready to be unleashed in the synthesis of adenosine triphosphate; and that is all that is happening- that is it.2089

This is a complete discussion of oxidative phosphorylation.2142

Once again, high-energy electrons, as they pass through the electron transport chain, they give up all of that energy.2146

The body uses that energy to pump protons and to create a proton gradient.2152

Now that that proton gradient is created, it has taken that energy, and now, it is potential energy.2158

Now, when protons fall back down their gradient going back into the matrix from the inter-membrane space, we are going to use that 400kJ to actually create ATP molecules that are going to be pumped out.2164

The body is going to use them however they see fit- that is it.2177

That is oxidative phosphorylation; thank you so much for joining us here at

We will see you next time, bye-bye.2185