From Photosynthesis to Respiration: Structure and Function of Energy Transforming Membrane Protein Complexes
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00:00
ChemistryPhotosynthesisMultiprotein complexCell membraneProteinMachinabilityReaction mechanismComputer animationMeeting/InterviewChemical experiment
00:45
NobeliumChemistryJohann Sebastian BachPhotosynthesisChemistProteinCell membraneSeparation processLecture/ConferenceMeeting/InterviewChemical experiment
01:20
PhotosynthesisMultiprotein complexEtherChemistJohann Sebastian BachChemistryVincristineProteinCell membraneCrystallographyThermoformingErdrutschSystemic therapyBioenergeticsMeeting/InterviewChemical experiment
01:55
ChemistPhotosynthesisMultiprotein complexMachinabilityPhotosynthesisWalkingNobeliumPhotosynthesisElektronentransferHarvester (forestry)Chemical reactionCell membraneQuinoneElectron donorMultiprotein complexMoleculeElectronElektronenakzeptorChemical experimentMeeting/Interview
02:50
ChemistPhotosynthesisEtherJohann Sebastian BachNobeliumHydrochinonElectronProtonationCell membraneCytochromeMultiprotein complexMeeting/InterviewChemical experiment
03:25
ChemistEtherPhotosynthesisJohann Sebastian BachTeilentrahmte MilchVolumetric flow rateElectronCell membraneElectron donorMultiprotein complexCytochromeProtonationLactitolElektrochemische ReaktionRespiration (physiology)Chemical experimentMeeting/Interview
04:20
Johann Sebastian BachMagmaPhotosynthesisChemistEtherChemistryOptische AktivitätSolubilityProtonationProtein subunitHydroxybuttersäure <gamma->PhosphateMultiprotein complexMeeting/InterviewChemical experiment
04:55
Multiprotein complexJohann Sebastian BachHarvester (forestry)Multiprotein complexMoleculeSeleniteSetzen <Verfahrenstechnik>Functional groupCarotinoideSpeciesCobaltoxideRadiation damageChemical experimentMeeting/Interview
05:50
PhotosynthesisEtherJohann Sebastian BachChemistMultiprotein complexChemistryCobaltoxideChemical reactionMoleculeExciter (effect)Cell membraneAlpha particleHelicität <Chemie>Meeting/InterviewChemical experiment
06:25
PhotosynthesisEtherChemistJohann Sebastian BachMan pageHarvester (forestry)Chemical reactionMultiprotein complexElectron donorCell membraneChemical experimentMeeting/Interview
07:20
PhotosynthesisJohann Sebastian BachEtherNobeliumChemistChemistryElektronentransferCarbon (fiber)Electronic cigarettePhysical chemistryChemical reactionNitrogen fixationAcidFoodKohlenhydratchemieGlykolyseStuffingMeeting/InterviewChemical experiment
07:55
Highway Addressable Remote Transducer ProtocolPhotosynthesisChemistJohann Sebastian BachMultiprotein complexThermoformingChain (unit)HydrogenMitochondrionWine tasting descriptorsChemical experimentMeeting/Interview
08:50
ChemistPhotosynthesisJohann Sebastian BachMultiprotein complexEtherAreaAcidThermoformingChemical reactionHydrogenCobaltoxideWaterWine tasting descriptorsChain (unit)ExplosionPhotosynthesisMeeting/InterviewChemical experiment
09:25
EtherChemistPhotosynthesisProtonationQuinoneMoleculeAddition reactionChemical reactionMultiprotein complexElectronReducing agentHydrochinonCell membraneCytochromeCobaltoxideCytochrom cShuttle-VektorWaterChemical experimentMeeting/Interview
10:20
ChemistPhotosynthesisJohann Sebastian BachAreaMultiprotein complexNobeliumNobeliumOctane ratingErdrutschChemical reactionCytochrom cMeeting/InterviewChemical experiment
10:55
Multiprotein complexChemistryPhotosynthesisReducing agentElectronWursthülleEnzymeAbbruchreaktionInorganic chemistryCytochrom cCopperCobaltoxideIronChemical experimentMeeting/Interview
11:50
Johann Sebastian BachPhotosynthesisChemistryChemical reactionMultiprotein complexChromerzChemistWaterProtonationElectronCell membraneSetzen <Verfahrenstechnik>Addition reactionYield (engineering)CytochromeMeeting/InterviewChemical experiment
12:25
Johann Sebastian BachMultiprotein complexNobeliumChemistEtherPhotosynthesisWine tasting descriptorsProton pumpCytochrom cReaction mechanismMultiprotein complexEnzymeThermoformingCrystallographyElectronic cigaretteLaundry detergentCell membraneMicelleChemical experimentMeeting/Interview
13:20
Johann Sebastian BachChemistPhotosynthesisEtherMonoclonal antibodiesImmunologyCancerCell (biology)HybridomtechnikCytochromoxidaseGenetic engineeringAntibodies (film)Meeting/InterviewChemical experiment
13:55
Multiprotein complexChemistryEtherJohann Sebastian BachChemistUranhexafluoridAntibodies (film)GeneMultiprotein complexCytochromoxidaseCrystallographyCollectingStarvation responseChemical experimentMeeting/Interview
14:50
ChemistNobeliumPhotosynthesisEtherJohann Sebastian BachChemistryMagmaMinimale HemmkonzentrationMultiprotein complexRadioactive decayIntergranular corrosionAntibodies (film)ProteinCytochromoxidaseAddition reactionMeeting/InterviewChemical experiment
15:25
Multiprotein complexCytochromeChemistJohann Sebastian BachPhotosynthesisEtherNobeliumAntibodies (film)Laundry detergentOctane ratingProteinMicelleMultiprotein complexCell membraneProtein subunitChemistryCopperAtomic numberChemical experimentMeeting/Interview
16:20
ChemistMagmaJohann Sebastian BachPhotosynthesisCureMultiprotein complexHydroxybuttersäure <gamma->ProtonationElektronentransferCopperEnzymeElectronCytochrom cCell membraneMeeting/Interview
16:55
CytochromeMultiprotein complexHydroxybuttersäure <gamma->PhotosynthesisJohann Sebastian BachChemistCeriumAmino acidLysineResidue (chemistry)Glutamic acidElectronAspartic acidArginineProtonationWasserbeständigkeitCell membraneGlutaminstoffwechselActive siteChemical experimentMeeting/Interview
17:50
Johann Sebastian BachChemistryPhotosynthesisChemistMultiprotein complexCytochromeEtherSieveEnzymeResidue (chemistry)ProtonationElektronentransferLysineFunctional groupLactitolActive siteCell membraneMeeting/InterviewChemical experiment
18:25
Multiprotein complexJohann Sebastian BachPhotosynthesisUreaAlpha particleProtein subunitHelicität <Chemie>LipideMoleculeResidue (chemistry)GeneEnzymeCell cycleCytochromoxidaseProtonationChemical experimentMeeting/Interview
19:20
Johann Sebastian BachPhotosynthesisChemistEtherPerm (hairstyle)Synthetic oilChemical reactionNobeliumMultiprotein complexChemistryCobaltoxideProtonationReducing agentProteinProtein subunitN-terminusC-terminusHelicität <Chemie>Meeting/InterviewChemical experiment
19:55
Multiprotein complexCytochromePhotosynthesisNobeliumChemistRNACopper proteinsAtomic numberWeinfehlerSetzen <Verfahrenstechnik>Starvation responseCopperElektronentransferAtomProtein subunitChemical experimentMeeting/Interview
20:50
PhotosynthesisMultiprotein complexChemistryJohann Sebastian BachHelicität <Chemie>Gas cylinderActivity (UML)ProtonationMeeting/InterviewChemical experiment
21:25
Multiprotein complexVimentinChemistEtherJohann Sebastian BachPhotosynthesisNobeliumTable wineHelicität <Chemie>Blue cheeseHelixProtonationCell membraneIon channelThermoformingElektronentransferChemical experimentMeeting/Interview
22:20
ChemistEtherPhotosynthesisSynthetic oilJohann Sebastian BachMinimale HemmkonzentrationMultiprotein complexHydroxybuttersäure <gamma->Ion channelCopperProtonationCobaltoxideMeeting/InterviewChemical experiment
22:55
Multiprotein complexChemistEtherJohann Sebastian BachPhotosynthesisMagnetometerCytochromeElectronProtonationIon channelElektronenakzeptorCytochromoxidaseProtein subunitAreaBeta sheetCopperChemical experimentMeeting/Interview
23:50
PhotosynthesisChemistryMultiprotein complexIon channelCell membraneCobaltoxideActivity (UML)Animal trappingSense DistrictSunscreenMeeting/InterviewChemical experiment
24:50
PhotosynthesisValineCopperElectronic cigaretteCobaltoxideWasserbeständigkeitPhenyl groupTryptophanIon channelLigandEnzymeMutageneseReducing agentCell cycleSense DistrictSunscreen
25:50
PhotosynthesisMultiprotein complexChemistIon channelCobaltoxideSense DistrictEnzymeActive siteCHARGE syndromeSide chainIonenbindungNeutralization (chemistry)PropionateGeneHydrogen bondFunctional group
26:50
ChemistPhotosynthesisMultiprotein complexLigandProtonationMutageneseHistidineCopperCell cycleThermoformingImidazolEnzyme
27:50
ChemistPhotosynthesisProtonationReaction mechanismLigandHistidineProton pumpCopperRedoxCrystalEnzymeOrigin of replicationArtifact (archaeology)Protein subunit
28:50
ChemistPhotosynthesisAmineFactor XIIIProteinAtomic numberThermoformingWaterMoleculeEnzyme
29:50
ChemistReaction mechanismAtomThermoformingLigandIronProton pumpHydroxylAtomic numberX-ray crystallography
30:50
Johann Sebastian BachPhotosynthesisChemistryNobeliumAtomic numberReducing agentReaction mechanismElectronThermoformingSense DistrictEnzymeReaktionsgleichungCHARGE syndromeMeeting/InterviewLecture/Conference
31:25
MitosisChemistryNobeliumJohann Sebastian BachPhotosynthesisEtherHighway Addressable Remote Transducer ProtocolDeterrence (legal)Multiprotein complexGetterCHARGE syndromeProtonationReducing agentElectronImidazolThermoformingLigandCobaltoxideBinding energyWaterMoleculeLecture/ConferenceMeeting/Interview
32:15
Johann Sebastian BachPhotosynthesisChemistNobeliumLaxativeAmalgam (chemistry)ProtonationWaterCHARGE syndromeReaction mechanismIon transporterHistidineLecture/ConferenceMeeting/Interview
32:50
PhotosynthesisNobeliumChemistMagmaEtherThermoformingHistidineAddition reactionProtonationReaction mechanismActive siteLigandCobaltoxideSpeciesEnzymeMeeting/InterviewLecture/Conference
33:25
AcetateTrihalomethanePhotosynthesisChemistryNobeliumJohann Sebastian BachChemistHydroxybuttersäure <gamma->EtherTiermodellIronHistidineLigandCopperLecture/ConferenceMeeting/Interview
34:01
Multiprotein complexNobeliumHighway Addressable Remote Transducer ProtocolChemistryAreaPhotosynthesisChromerzHistidineTyrosinPosttranslational modificationNucleic acid sequenceDNS-SyntheseChemical reactionElectronPotenz <Homöopathie>Chemical experimentMeeting/InterviewLecture/Conference
34:45
NobeliumJohann Sebastian BachChemical reactionEtherChemistPhotosynthesisMultiprotein complexTyrosinReaction mechanismRadical (chemistry)Cross-linkHistidineCystinPotenz <Homöopathie>Setzen <Verfahrenstechnik>PeroxidaseChemical reactionProteinLecture/ConferenceMeeting/InterviewChemical experiment
35:20
ChemistPhotosynthesisJohann Sebastian BachEtherSauerrahmCytochromoxidaseIronProtonationCopperThermoformingElectronCobaltoxideChemical compoundSetzen <Verfahrenstechnik>Meeting/InterviewChemical experimentLecture/Conference
35:55
Multiprotein complexCytochromePhotosynthesisChemistryEtherJohann Sebastian BachUranhexafluoridThermoformingIronChemical compoundPeroxideElectronCopperMetalCobaltoxideMeeting/InterviewChemical experimentLecture/Conference
36:31
PhotosynthesisJohann Sebastian BachMultiprotein complexCobaltoxideResonance (chemistry)Chemical compoundIronElectronTyrosinResidue (chemistry)Chemical experimentMeeting/InterviewLecture/Conference
37:15
PhotosynthesisNobeliumChemistCopperIronTyrosinElectronRadical (chemistry)Lecture/ConferenceMeeting/InterviewChemical experiment
37:50
PhotosynthesisEtherNobeliumChemistryJohann Sebastian BachChemistProtonationÜbergangszustandData conversionReaction mechanismMeeting/InterviewChemical experimentLecture/Conference
38:25
Multiprotein complexMortality rateNobeliumChemistJohann Sebastian BachPhotosynthesisHighway Addressable Remote Transducer ProtocolChemistryWaterSetzen <Verfahrenstechnik>ProtonationMoleculeReducing agentPropionsäureMeeting/InterviewChemical experimentLecture/Conference
39:01
Multiprotein complexNobeliumJohann Sebastian BachChemistProtonationRapidAreaReaction mechanismProteinAspartic acidElectronic cigaretteMetabolic pathwayLysineGlutamic acidElektronentransferChemical experimentMeeting/InterviewLecture/Conference
39:45
PhotosynthesisGlutamic acidPropionsäureElectronIon transporterCobaltoxideAreaLecture/ConferenceMeeting/InterviewChemical experiment
40:20
ChemistryJohann Sebastian BachChemistMultiprotein complexNobeliumKohlenstoff-14Precursor (chemistry)Infrarot-SpektrumReducing agentCytochromoxidaseMeeting/InterviewChemical experimentLecture/Conference
40:55
EtherChemistJohann Sebastian BachPhotosynthesisCoordination numberElectronic cigarettePropionsäureReaction mechanismReducing agentConformational isomerismMeeting/InterviewChemical experimentLecture/Conference
41:31
Johann Sebastian BachPhotosynthesisElectronic cigaretteAntibodies (film)ProteinChemical experimentMeeting/InterviewLecture/Conference
42:15
NobeliumPhotosynthesisChemistCalculus (medicine)Infrarot-SpektrumBiochemistryLecture/ConferenceMeeting/InterviewChemical experiment
42:51
Nobelium
Transcript: English(auto-generated)
00:15
So, thank you. As indeed was said, I am going to talk about membrane proteins.
00:22
You will see lots of membrane protein structures, but I should make clear from the very beginning that we don't do membrane protein structures in order to see how the structures look like. We would like to know how these machines work, and for this we have to know what the structure is, and this will then form the basis to understand the mechanism of
00:44
action of these molecular machines. And I should say that membrane proteins in general perform many important roles, but the major problem is that we cannot study them in great detail because we don't have much membrane protein, much of the membrane protein available from the amount, and also
01:05
they are very difficult to handle and we have still a severe lack of knowledge about membrane proteins. Membrane proteins constitute about 40% of all proteins of your body, but we know at present about 20 membrane protein structures from 12 different proteins, and we know about
01:25
maybe 5,000, 7,000 water-soluble proteins. And this tells you where the challenges are, and the challenges in membrane proteins are primarily crystallization. Crystallography is a method with many different methods. And one of the points of my lecture will be that you need many different methods nowadays
01:46
in order to solve biological problems. And the first slide I show you the bioenergetic system from pergore bacteria which is a form of photosynthesis.
02:02
And we have here in the heart the blue machine here is a photosynthetic reaction center and that's the one we were awarded the Nobel Prize in 1998. And the next step is, and this actually, this machine gets the energy from the light
02:20
harvesting complexes, you see here this one in pink, and which surrounds the whole reaction center and actually there are more light harvesting complexes which were in the membrane which transfer the energy from the light harvesting complex two to the light harvesting complex one to the reaction center where we get electron transfer.
02:41
I will only shortly touch the reaction center. The role of the reaction center is to transfer electrons from a primary electron donor here across the membrane towards acceptor molecules which are quinones here with a QB and we have to transfer a second electron to get a double protonation.
03:05
We have the hydroquinone formed. The hydroquinone diffuses into what's in the membrane towards the cytochrome PC1 complex, gets oxidized there and the protons are transported across the membrane in this complex.
03:22
And the electrons in the cytochrome C2 diffuse back in the periblasmic space of the bacterium and reduce the primary electron donor again via bound heme groups. So we have a cyclic flow of electrons and the purpose for that cyclic flow of electrons
03:44
is to pump protons in the cytochrome PC1 complex across the membrane. And we will hear more about the cytochrome PC1 complex in the following talk given by Hans Deisenhofer. And the cytochrome PC1 complex also plays a very important role in respiration and
04:03
you will see this complex on a similar scheme. The electrochemical proton gradient consisting of an electric field which is the more important common component and a proton gradient drives protons back through the membrane and it's
04:21
now clear from the work of Paul Breuer who presented that part yesterday and John Walker and his colleagues in Cambridge that you have here this water soluble complex for which they determine the structure that the gamma subunit here rotates and the backflow
04:42
of protons here drives the rotation of the gamma subunit and per one rotation of the gamma subunit you get one ATP formed from ATP and phosphate. ATP is the general currency of life, you could call it the euro of life.
05:04
So we have already a unified energy currency in biology, we will have it in Europe maybe in the world at the end also. Now I start with the light harvesting complex. You see here the structure of a light harvesting complex determined by us recently in Frankfurt
05:22
and you see here in green chlorophyll molecules. We have two rows of chlorophyll molecules, a lower row, there are eight chlorophylls in this complex, there are 16 of this type up here and they are actually linked, they are bridged by carotenoid molecules. Carotenoids are very important molecules in life.
05:43
First, they have a photoprotective function and they prevent the photo damaging effect of activated oxygen species and also they quench triplet states of chlorophylls which could generate such excited oxygen molecules.
06:02
And second, they absorb light and they transfer the energy to the chlorophylls and the chlorophylls convert and transfer the energy to the next light harvesting complex and then to the reaction center where some work is done. This here is the same complex viewed from the top onto the membrane.
06:24
You see here two helical membrane proteins, this is so called alpha subunit, this is the beta subunit and in between we have in green the chlorophylls, one circle and another chlorophyll we have here and we have you see again here the caroteneids with their
06:41
double role. And this kind of circular arrangement is quite remarkable and maybe nature invented some kind of synchrotron before man did it. So nature maybe is always first. And how the whole structure is arranged is the membrane, photostatic membrane is in here
07:02
We have here the light harvesting complex two, that's the light harvesting complex one which surrounds the reaction center, you see here in yellow the reaction center and the energy, it's only the energy is transferred from here to the next and from here to the primary donor of the reaction center where you see the respective chlorophylls here
07:24
vaguely in red and there you get electron transfer. And electron transfer then is chemistry, we start off with physics light absorption energy transfer and then electron transfer which is chemistry. And at the end we generate ATP, the ATP is used to fix carbon dioxide, we synthesize
07:42
carbohydrates in the dark reaction and this is the food where we all live from. And of course we eat the food, we degrade food and at the end we have the cytic acid cycle, we have glycolysis and we end up in the splitting of our food stuff into hydrogen, in some biological form of hydrogen and which is then converted further.
08:08
And this is done in the respiratory chain. And the respiratory chain is shown on this slide, you see here the respiratory chain
08:22
of a bacterium called paracoagustin hardrificans which is quite well studied, it has the advantage that you can use genetic methods in order to study the role of these components and also it appears to be closely related to the ancestor of your own
08:41
mitochondria and everything what I tell you here is also valid for your own mitochondria in your own body. First you generate NADH in the cytic acid cycle and this is the bound form of hydrogen in biology and what the respiratory chain does it, it converts it
09:05
with oxygen to form water and it has developed a quite complicated machinery not only to prevent that you have a detonating gas reaction to prevent explosions in your body all over and the loss of energy and also you like to
09:22
make use of the energy and the principle is the same as in photosynthesis. We have here four complexes. NADH is first oxidized here in this complex called complex one and protons are translocated across the membrane in addition to some electrons to reduction
09:43
of the of quinone molecules so we reduce quinone molecules in the reaction center, complex one does the same, the hydroquinone diffuses in the membrane towards the cytochrome BC1 complex and we will hear all the details of that complex in the subsequent talk.
10:02
Electrons there are transferred towards cytochrome C, cytochrome C diffuses towards the cytochrome C oxidase, this is this complex and this complex is of particular interest because it is the one where oxygen is reduced, water is formed and here we have some kind of vectorial reaction, this kind of
10:26
vectorial reactions were first described by Peter Mitchell in his so-called chemiosmotic hypothesis for which he was awarded the Nobel Prize 2 and this is a very important concept and it was very tough to get the concept
10:42
brought through and he thought about 20 years and I think that Boyer yesterday had a slide where he showed you the rate of acceptance of his hypothesis among his colleagues but I think now nearly everybody accepts it and cytochrome C
11:01
oxidase there is the terminal enzyme so electrons from cytochrome C are transferred to a binuclear copper A center so we have here some inorganic chemistry going on, reduction of two copper atoms, electrons are transferred further to a first heme A molecule, heme A, which is in this case
11:27
called simply heme A and the electron further transferred to a second heme A which is now called heme A3 for historical reasons and pretty near
11:40
the heme A3 iron we have a copper B bound and the active site, the iron and the copper B, that's the place where oxygen is bound, where electron reduce the oxygen, where protons are taken up, protons are taken up
12:01
exclusively from the inside and water is formed so we get creation of electric field by having the electrons from the outside and the protons from the inside and in addition nature has invented a trick to transport the same amount of protons as they are consumed by water formation across the membrane
12:23
and this doubles the energy yield in our cytochrome C oxidase. The consequences of course that you need only half of the foot in order to get the same amount of ATP at the end. Nowadays of course in Europe we have the opposite problem and there are certainly are people which would like to switch
12:41
off the proton pump in order to have a less efficient energy conversion and to fight obesity in their body. But certainly this is not the approach, not the goal of our research to abolish the proton pump inside the cytochrome C oxidase. We tried again a method of crystallography to get the
13:03
structural information which you need to understand the mechanism of this enzyme at the end so we tried again to crystallize it, isolating this complex first from the membrane in the form of detergent micelles and trying to crystallize this didn't work from the beginning and we used another
13:22
trick. We used monoclonal antibodies so we use methods from immunology in order to get crystals. We use methods from gene technology to produce crystals. We produce monoclonal antibodies first which means we take a mouse, immunize it with this complex. The mouse develops antibodies against
13:42
the cytochrome C oxidase. We sacrifice the mouse, take the spleen cells, fuse them with cancer cell lines, get a hybridoma cell lines which produces antibodies against the cytochrome C oxidase. Then we continue, isolate the genes for these antibodies and express these genes in bacterium E. coli.
14:04
Then we go on in our stuff, make the complex of cytochrome C oxidase and a part of the antibody fragment and crystallize this complex and these are now the crystals. They have a length of about one millimeter, diameter is about 0.3. Then we use crystallography and the most important point when you get
14:25
crystals is that they should diffract electrons well. The diffraction here was I would say was moderate at best. You see here diffraction pattern taken at a synchrotron and the synchrotron actually was in Japan in Scuba and this
14:41
synchrotron was particularly helpful because it had a mode of data collection which could be used for less well diffracting crystals and also for radiation sensitive crystals much better than the European ones. And the diffraction limit here is about 2.8 angstrom but perpendicular to it
15:01
it's only about 4 angstrom which is just the limit of getting a protein structure. Nevertheless you see here now the structure of the entire complex so this is the cytochrome C oxidase. This is the antibody fragment which we used for getting the crystals. It helps to form the
15:23
crystal lattice and without this addition to what cytochrome C oxidase we would not get crystals because most of the protein here is actually buried in a detergent micelle and in a detergent micelle this part of the protein is not available for crystallization. So this is why we had to use this antibody
15:41
fragment and we tried it also with the PC1 complex. It worked well and at present the method we have a 100% success rate and I hope that we will be able to determine more membrane protein structure in the near future. And I hope you can hear about more membrane proteins three years from now in the same chemistry symposium here.
16:07
Here you see here in purple subunit 2. You barely can see here copper atoms bound to the protein. You can see here in red now a heme A
16:28
and here is the copper B. So electron transfer is restricted to this rather narrow part here. Cytochrome C binds towards this corner of the enzyme and transfers the
16:43
electron towards here and then electrons transferred here and then here. Protons are taken up from the inside and protons are pumped across the membrane and it is of great interest to understand how the transfer of electrons and protons are coupled. What's also of particular interest is the
17:05
distribution of amino acid residues and I show you here the distribution of those residues which are frequently charged in nature. Of course arginine and lysine can be positively charged. Aspartic acid and glutamic acid
17:23
can be negatively charged and we have only very few in the hydrophobic environment of the membrane and they are there for either structural or functional reasons. These are for structural reasons. This is there, we don't know it yet for structural reasons. This is here, a glutamic acid residue and when you
17:43
change this by genetic methods, so you have to use genetic methods to site direct immunogenesis to change it towards even to a good glutamine which is a rather minor change. The enzyme is dead. It doesn't work and this is therefore of great importance. There is another residue of great importance
18:03
which is this lysine here in blue and if you change this to a neutral residue, you get the same phenotype. The enzyme is dead. It doesn't function and we presume that these residues are involved in proton transfer towards the active site and this may be also involved in transferring
18:24
those electrons, those protons which are transported across the membrane and I will also discuss some more residues later on. I simply now describe individual protein subunits. We have here subunit 3. Subunit 3 has an open v-shaped
18:45
arrangement. We have two transmembrane alpha helices here. We have here five transmembrane alpha helices and we have some bound lipid molecules here in the cleft and the role of subunit 3 is not well understood. There are two suggestions.
19:03
First, you can delete the gene in our bacterium and what you get is you get a reduced amount of cytochrome c oxidase consisting of two subunits. These two subunits cytochrome c oxidase is still active in proton pumping and in the turnover of the enzyme so it can have nothing to do with the subunit
19:23
with proton pumping or with the oxygen reduction. We think actually that this subunit has a role in oxygen diffusion and I will talk about this point later on a little bit. Here I show you subunit 2. Subunit 2 has an N-terminus
19:46
up here pretty close to the C-terminus. We have an irregular protein fault. Then we have two membrane-spanning helices. Then we have through here a kind of protein fault which was already known. It corresponds to that of the type 1 copper
20:04
proteins and there are some differences. Primarily, we have here we have two copper atoms. You see them here at the tip together with the mode of finding. I don't go into further details due to the time limits and why we have
20:20
here two instead of only one copper atoms is not well understood. It might be due to the reorganizational energy and what this actually is probably we will hear tomorrow by Rudy Marcus and he would develop the theory of electron transfer and the reorganizational energy is certainly an important parameter and
20:42
might be the reason why we here have two and not only one copper atom. This is here subunit 1. Subunit 1 has a rather regular appearance. Looks like a cylinder consisting of 12 transmembrane helices and in here we have the binding
21:00
side of the prophyrin rings, the hemes. We have here the heme A3 and quite interesting the hydrophobic tail here is bent away and this allows the excess of protons towards the active side here. And I will discuss this point also
21:22
further and here you see subunit 1. Now from the top and this is actually the most interesting view. You see the 12 transmembrane helices. They are all tilted. They are rather long, much longer than was expected. Here we have helix number 1, the
21:41
brown one. Number 2 is the green one. Number 3, the blue. 4 is purple. 5 is the red kinked. This red one is 6. You continue like this with counting. It's a sequential, simple topologies, sequential arrangement of helices. However, 4 helices
22:00
each in projection form a half circle. So here we have one half circle. Here we have another half circle. And here we have the third half circle. And in this kind of arrangement you generate three pores. So we have here some kind of pore and we
22:20
think that this pore is used for proton transfer to the center of the membrane. And then the proton is diverted towards the active side. Here we have the second pore. And this pore is used for proton access towards the active side, partly.
22:41
Controlled proton access to the active side. And then the pore is blocked by the heme A3, which is seen edge-on. Here you see the copper B nearby. And here's the place where the oxygen is bound. And the tail here is bent away again, as I
23:01
mentioned already, in order to allow access of protons towards this place. Here we have the third pore and this appears to be tightly blocked by the heme A, which is the first electron acceptor which transfers the electron towards this other heme. The entire cytochrome C oxidase can be seen here from the
23:23
terryplasmic space again in a simplified truncated version. Subunit 1 in yellow, subunit 2 in blue, together with a beta strength-rich area. And you can see here this part covering here additionally the heme A3 and the copper B, which is in this position.
23:47
And you see also very nicely here subunit 3. And what we actually think is we have identified a hydrophobic channel from the binding side of the lipids towards the active side here. And we think that this channel actually is used for the
24:06
diffusion of oxygen towards the active side. And you are probably not aware of the fact that oxygen is a hydrophobic entity. It likes to be enriched in membranes and you know from spectroscopy that it is enriched in the
24:22
membrane by a factor of 7. And therefore it's very likely and makes good sense if we have oxygen diffusion from the lipidic milieu from the membrane towards the active side. And if we here have loosely bound lipid molecules, we are able probably to enrich oxygen here further. So this may be an oxygen trap and the
24:44
oxygen then can diffuse from this oxygen pool towards the active side. Here you see more details of this oxygen diffusion channel, of this presumed oxygen diffusion channel I should say. You see primarily
25:02
hydrophobic residues, phenyl rings lining up, tryptophan residues, and at the end you have a valine. Here we have the binding side for the copper B together with a ligand. Here we have the heme A3. And it is known from spectroscopy that when oxygen diffuses in, it first becomes temporarily bound towards copper B and
25:25
then it is transferred to the heme A3. This also makes sense from the channel structure. And side-directed mutagenesis genetic method has shown that when you change this valine to the slightly larger isoleucine, you get a reduction of the
25:43
diffusion speed, which means that the Km value for the enzyme for oxygen is increased by a factor of 10, but the maximum turnover number of the enzyme is still the same if you simply have a 10 for higher oxygen concentration. So this makes sense with the assignment of this channel to
26:04
an oxygen diffusion channel. Now I come towards the most complicated thing in my talk. This is the environment of the binding site of the heme A3. You see the heme A3
26:20
light blue together with a protein. The heme groups have two propionate side chains. So here we have a propionate side chain and this is charged, neutralized by forming an ion pair with an R gene in here. We have the second
26:41
propionate here and this is not charged neutralized. The charge there gets stabilized by accepting hydrogen bonds from neighboring residues here. Quite interestingly, we also had from the very beginning, we could identify the copper B down here and from side-directed mutagenesis work, it has been
27:03
known that there are most likely three histidine ligands, but we could only identify two histidine ligands and the other one for the third one. We had no electron density at all and there had been the proposal in the literature that a histidine might change its protonation state during the turnover of the
27:26
enzyme being negatively charged, meaning imidazolate form in one state of the enzyme becoming imidazole and imidazoleum, the positively charged form in the enzyme and the imidazoleum form cannot be, cannot be ligand to
27:44
copper B. So the histidine could shuttle between two positions depending on the protonation state and it could carry over protons by switching between two, the two positions and this might be the mechanism of how a proton pump works. But this, I have to say, is speculation and there is at present no
28:03
evidence for that. And when we repeated the whole experiment in the absence of acite with oxidized and reduced forms, we could not discover the absence of the histidine ligand. We could clearly see the ligand of the histidine as being still a ligand to
28:23
copper B and it stays there upon oxidation and reduction of the enzyme and we stop when we think that the absence of the histidine ligand was an artifact due to the presence of acite. So you have to be pretty careful. As I mentioned, we had some problems with our original crystal form and a graduate
28:44
student in the lab, Christian Ostermeier, who also had made the first crystal form, worked very hard to get better crystals. He succeeded. Now with the two subunit form of the enzyme, only two proteins of unit presence, he got these crystals. They don't look as nice as the other ones. The protein is pretty unhappy. You
29:04
see some teenager protein under the crystallization conditions, but the crystals are much better than our first version. So we could determine an improved structure and the improvement was largely we had a much better definition of the position of the atoms and we could start to identify bound to
29:25
water molecules and each of these green ball means the position of a water molecule and much to my surprise you see many water molecules up in the upper half of the enzyme but only very few below them, which probably means
29:44
that we have also water molecules here but they are much less ordered whereas the water molecules here are much better ordered, much more highly ordered and this could have some functional significance. This here, coming back to mechanism, shows you the electron density around the kappa v. This is the
30:03
kappa v electron density and this is the missing electron density for a histine ligand and this could be interpreted in the form of a mechanism of proton pumping. When you consider that you actually have an iron atom here, a
30:24
kappa v atom, the iron has a formal charge of plus three, the kappa of plus two and you most likely have an OH minus, a hydroxy group between both atoms cannot be resolved by x-ray crystallography at our resolution but there is
30:41
spectroscopic evidence for that and I think also otherwise if you would have only the positive charges here, you would get a two strong electrostatic repulsion, the distance is only about five angstrom and the whole thing nearly would explode if you don't wouldn't have a negative charge between both atoms so the OH here makes good sense. The basic idea however in this histine cycle
31:04
mechanism is when you start from the oxidized form of the enzyme, you get a reduction of the enzyme from the periplasmic base, the electron is transferred first to the iron, then it hops over, jumps over onto the kappa and this then disturbs the charge balance in the whole environment and in order to
31:26
regain the charge balance, you take up a proton very simply and this then converts upon the first reduction this kappa b is ligand from the imidazolate to the imidazole, then you take up the second electron
31:44
which stays on the iron, you neutralize this additional charge by taking up a proton, you convert this ligand to the imidazolian form and the imidazolium can no longer be a kappa b ligand and it flips over in this position. Then you bind oxygen. This is known that only after full
32:01
reduction you bind oxygen here and then the chemistry, if the oxygen starts you get uptake of protons to form the first water molecule. When you take up protons, you disturb again the charge environment and the charge of the protons then expels these two protons up here and so the uptake of the two
32:24
protons to form water would expel the protons already here in order to have the same charge environment. And this hypothesis I think is the most simple one for explaining a pump mechanism but certainly cannot be
32:40
phrased in the term of a histidine cycle and we have to think of alternatives. This is now the electron density for the oxidized and reduced form and in the histidine cycle in the reduced form we should not have all the histidine ligands present. They are there and this is I think is one of the fundamental experiments which tells you that the histidine cycle mechanisms
33:04
unlikely. In addition I have great problems to see how a positively charged histidine would not deliver back its proton towards a reduced oxygen species at the binuclear site. Now I show you some more interesting new details.
33:21
Again it's the heart of the enzyme. You see an unbiased electron density map calculated by a technique called simulated annealing or midmap. You omit from the model all these residues and start to and do some simulated annealing around in order to get a way of bias. Use the new phases, calculate an
33:44
unbiased electron density map and this is this electron density here and you rebuild the model. You see here the model of the EMA3. This is the iron. It's a histidine ligand to the iron. You see here the copper B and you see here the histidine ligands towards copper B. This is the third one and much
34:06
to the general surprise what you find is that a nearby tyrosine is covalently cross-linked with a histidine. So we have here an unexpected covalent cross-link and of course this kind of thing, this
34:23
modifications cannot be discovered from DNA sequences. You still have to do some further experiment apart from DNA sequencing and there is some meaning about this and I would think that the principal meaning is that you get such a high oxidative power during the reaction that it extracts an
34:44
electron from the environment. You generate a radical and it's known that tyrosine can form this kind of radicals and then in a radical mechanism you simply get this kind of cross-link and such kind of cross-links also have been observed not with histidines but with, for instance, cystines
35:03
and other residues in peroxidases. So peroxidases in general have enough oxidative power to generate radicals which then leads to cross-links in proteins and this means we can have the same type of reaction here. Now I
35:20
think becomes more systematic. This is now the catalytic cycle of cytochrome c oxidase and this is simple. We start off with oxygen. The iron is in plus three form, copper plus two. Then we put on the first electron which goes onto the copper converting onto copper one and this is known that
35:40
this is accompanied by the uptake of a proton. This is the one electron reduced form. Take up the second electron accompanied by the uptake of a proton, you get the reduced form iron plus two, copper plus one. Then you take up the oxygen only and you get formation of the so-called compound A
36:02
which was discovered by Pritan chance in Philadelphia more than about 20 years ago and then you form a compound called P. P was for a long time thought to be a peroxy compound, at least a form of peroxy compound, iron plus three, copper plus two, but the electrons from the metals are
36:24
transferred onto the oxygen. I always consider this as a very unlikely structure and I wonder how this could be stable. And actually my skepticism was, I should say, was satisfied by a recent experiment in Japan by Kitagawa who by resonance Raman spectroscopy got clear-cut
36:43
evidence that you actually have already in the P state a split oxygen compound and you have actually an oxyferule. This means an oxygen bound to the iron in a double-pointed way, iron plus four, oxygen minus two in this form and
37:00
you have the copper plus two. But this would then mean that you miss an electron here and this compound has to steal an electron and the possibilities are that it's stolen, the electron is stolen from the porphyrin, it's stolen from the residue and the tyrosine is a good explanation. People discuss also that a copper might become copper plus three and also
37:24
people discuss that the iron might be plus five, but these are all less likely than this explanation where you steal the electron from a tyrosine creating a tyrosine radical. So, but this is under debate but I think that this route became much less likely within the last year.
37:44
So this is a rather new experiment. The major problem that we then have is however that it is known primarily from Lichtstrom's work in Helsinki that the proton pumping, sorry, proton pumping is of course only in the
38:02
transition from the P state to the F state. This state here is well characterized and everybody agrees that this is an oxophorite state here and it's also known that the next two protons are pumped in the conversion, in that way of conversion. And if we have this kind of mechanism we might, we should
38:24
have observed proton pumping already here and we have to think about a way out that we don't have proton formation of water molecules and the incoming protons expel the protons which have been taken up during a reduction at that time. So now I just want to come back towards the structure. You see here in red
38:46
together with the histine line is the heme A. This is the heme A3 and the point I want to discuss is where do the protons go which are taken up upon reduction.
39:01
I think actually that the protons which are taken up are stored on the propionates in this area up here which, and there appears to be a rapid equilibration of protons in that area and later you get uptake of the protons and I have formulated a mechanism which I think would be in agreement with all kind of mechanisms.
39:27
Actually there are, in the protein which I didn't show so far, there are two identifiable proton transfer pathways. This one with lysine involved and this one here which has an aspartic acid at the beginning and a
39:43
glutamic acid at the end and I think that the protons are delivered from this glutamic acid onto the propionates towards this area during the pumping. And when electrons come in towards forming the oxygen they are then expelled from that area to the outside. This would explain pumping. But I cannot go
40:01
further into details in order to explain that cycle and I would take too long now to explain these details and you have to contact me privately in order to discuss it further. But just to say we need different methods in order to prove this and what we did now, we made a biosynthetic efficient mutant for heme A
40:23
and fed isotopically labeled precursors in a way that only this carbon atoms are labeled with 13C of the heme A and did Fourier transform infrared spectroscopy. So we now use even another method and we look what changes in the
40:42
vibrational bands upon reduction and you see this for labeled and unlabeled you see the differences for labeled and unlabeled cytochrome C oxidase and there are clear differences which tell you that there are changes of coordination states and conformation of the propionates upon reduction and this
41:01
at the end can be summarized in some kind of mechanism which purely operates on electrostatic grounds and this is now our working hypothesis and we work very hard either to pre-proof or disprove such kind of mechanism in detail but at least you get an idea the whole thing is complicated and still
41:25
may have to get an idea because we have to go into details and we need all the methods and I think the message for you is for the students is that you have to know all the methods in order to use them efficiently to solve biological problems and the people at the end I would like
41:43
to acknowledge are here from my own group, Derek Lyman establishing the method of the antibody fragment production in the lab. Christian Ostermeier did most of the crystallization work and also determined the second crystal structure. Hannemiller isolated most of the material. So Iwata isolated in a
42:05
very, sorry, solved the protein structure in a very short time. Axel Harringer then improved his structure and got now a much better structure. Imo Kant did electrostatic calculations which, theoretical calculations which I
42:22
didn't mention and Julia Wehr is involved in this Fourier transform infrared study studies together with Werner Mengele, Peter Helwig from the University of Munich. Some mutant work was done at Frankfurt University by our biochemical collaborator Bernd Ludwig and Eike Witt. Now I thank you for your attention.