Biophysics of Mitochondria

205 Pages·2019·5.912 MB·English

by  Nikolai Vekshin

Nikolai Vekshin
Biophysics
of Mitochondria
Biophysics of Mitochondria
Nikolai Vekshin
Biophysics of Mitochondria
NikolaiVekshin
Moscow,Russia
Previouslypublishedwithotherpublisher.
ISBN978-3-030-33852-7 ISBN978-3-030-33853-4 (eBook)
https://doi.org/10.1007/978-3-030-33853-4
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Preface
Mitochondria are one of the most important organelles in aerobic cells. They have
been studied during about 100 years by cytologists, biochemists, molecular biolo-
gists,andbiophysicists.
The monograph summates a lot of important results on the investigation of
mitochondria using a variety of biophysical methods: fluorescence spectroscopy,
UV-vis spectrophotometry, IR spectroscopy, light scattering, optical microscopy,
fluorescence microscopy, colorimetry, photobleaching, polarography, pH-metry,
andsoon.Especially,methodologicalissuesareaccentedhere.
Aconsiderablepartofthebookisbasedonmyownlong-termexperience.Also,I
havepresentedmainexperimentaldataofmanyotherresearchers.Itriedtoanalyze
indepthalternativepointsofview,newhypothesis,andinterestingcontradictions.
Ihopethat thismonograph will beuseful forresearchersandgraduatestudents,
specializing in mitochondrial biophysics, as well as in biochemistry, molecular
biology,andcytology.
This book was published in Russian in 2019 by Photon Publishers (Pushchino,
Moscowregion).
I am very grateful to my colleagues and PhD students who participated in
conducting the experiments with mitochondria over long time, namely,
A.S. Lomtev, I.V. Sharova, A.N. Zinina, R.L. Anisimov, D.A. Shishmakov,
I.B. Sokolova, V.I. Sukharev, V.I. Kovalev, E.A. Begunova, D.N. Kurdyukov,
K.E. Krekhova, M.A. Frolova, A.V. Chaplygina, A.M. Lvov, and many others,
listedascoauthorsinjointpublications.
Moscow,Russia NikolaiVekshin
September2019
v
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 MythsandReality. . .. . . .. . . . .. . . .. . . .. . . . .. . . .. . . . .. 1
1.2 MitochondriainCell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 EarlyStudy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 MainPropertiesofMitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1 StructuralFeatures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 GiantMitochondriaandReticulum?. . . . . . . . . . . . . . . . . . . . . . 10
2.3 EvolutionaryOrigin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.4 ReproductionofMitochondriainCell. . . . . . . . . . . . . . . . . . . . 15
2.5 DoMitochondriaSwellMuch?. . . . . . . . . . . . . . . . . . . . . . . . . 18
2.6 MitochondrialDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.7 Membrane’sandMatrix’sProteins. . . . . . . . . . . . . . . . . . . . . . . 27
2.8 Bioenergetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3 StructureandFunctionofRespiratoryChain. . . . . . . . . . . . . . . . . . 35
3.1 RespiratoryChain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2 NADHDehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 SuccinateDehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4 CytochromeOxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4 Physico-ChemicalMechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1 InteractionsinEnzyme-SubstrateComplexes. . . . . . . . . . . . . . . 49
4.2 MechanismsofElectronTransport. . . . . . . . . . . . . . . . . . . . . . . 50
4.3 Membrane’sSynthesisofATP. .. . . . .. . . .. . . . .. . . .. . . . .. 52
4.4 ProtonTransportandChemiosmoticHypothesis. . . . . . . . . . . . . 54
4.5 OnMeasurementsofTransmembranePotential. . . . . . . . . . . . . . 56
4.6 ConformationalHypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
vii
viii Contents
4.7 ThermalModelofCoupling. . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
5 Photoreactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5.1 Photo-RespirationandPhoto-InducedATPSynthesis. . . . . . . . . 73
5.2 Photo-DesorptionfromSurface. . . . . . . . . . . . . . . . . . . . . . . . . 76
5.2.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.3 FlavinandUbiquinoneAreNotInvolvedintheTransfer
ofElectronstoArtificialAcceptors. . . . . . . . . . . . . . . . . . . . . . 80
5.3.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
6 Red-OxReactionswithNADHDehydrogenase. . . . . . . . . . . . . . . . . 87
6.1 Lag-PeriodandPseudo-OscillationsinNADHRed-Ox
ReactionswithDichlor-Phenolindophenol. . . . . . . . . . . . . . . . . 87
6.1.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.2 Electron-ConformationalPropertiesofFlavoprotein
FragmentofEnzyme. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.2.1 PolarizationandPhaseModulationSpectroscopy. . . . . . . 93
6.2.2 TryptophanFluorescenceDecay. . . . . . . . . . . . . . . . . . . 94
6.2.3 CorrelationConfocalMicroscopyofFlavins. . . . . . . . . . 95
6.2.4 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.3 ResonantEnergyTransferfromTryptophanstoNADH. . . . . . . . 96
6.3.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.4 LossofFlavinfromEnzyme. . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.4.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6.5 StabilizationofEnzymebyAdenosinePhosphates. . . . . . . . . . . 104
6.5.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
6.6 DehydrogenaseActivityofMitochondrialSmear,Detectedby
DecreaseofTryptophanFluorescencebyFormazan. . . . . . . . . . 111
6.6.1 NADH:pNTVReductaseActivity
intheMCSuspension. . . . . . . . . . . . . . . . . . . . . . . . . . 113
6.6.2 NADH:pNTVReductaseActivity,asDetermined
FluorimetricallyinaSmearofMitochondria. . . . . . . . . . 115
6.6.3 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
6.7 Rotenone-InsensitiveOxidationofNADHbyFragments
ofRespiratoryChain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6.7.1 OxidationofNADHinSuspension
ofCardiacMitochondria. . . . . . . . . . . . . . . . . . . . . . . . . 119
6.7.2 SDSEffect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.7.3 ObtainingFractionsofDetergentMembrane
Fragments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.7.4 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
6.8 CytochromeCShuntstheNADHOxidation. . . . . . . . . . . . . . . . 126
Contents ix
6.9 NADHOxidaseActivityofMitochondria
inHypotensionatBlockofRespiratoryChain. . . . . . . . . . . . . . 127
6.9.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
7 Proto-mitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.1 SomePropertiesofProto-mitochondria. . . . . . . . . . . . . . . . . . . 139
7.1.1 FractionationofaSuspensionofMitochondria
onFilters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.1.2 FluorescentProperties. . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.1.3 OxidationofNADH. . . .. . . . . . .. . . . . . . .. . . . . . . .. 143
7.1.4 EffectofSucroseonNADH:FerricyanideReaction. . . . . 145
7.1.5 ProteinProfile. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
7.2 Proto-mitochondriaofLiverCells. . . . . . . . . . . . . . . . . . . . . . . 146
7.3 GerminalProto-mitochondria. . . . . . . . . . . . . . . . . . .. . . . . . . . 154
7.4 FluorimetricComparisonofProto-mitochondria
andMitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.5 PhotometryandFluorimetryofProto-mitochondria
fromLiverofYoungandAdultRats. . . . . . . . . . . . . . . . . . . . . 170
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
8 DegradationofMitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
8.1 DegradationofMitochondriatoLipofuscinbyHeating
andUV-Lighting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
8.1.1 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
8.2 DisruptionofFlavinHomeostasisinIsolatedMitochondria. . . . . 184
8.2.1 ChangesinFlavinFluorescence. . . . . . . . . . . . . . . . . . . 186
8.2.2 Non-enzymaticHydrolysisofFADbyIronIons. . . . . . . 188
8.2.3 Non-enzymaticSuperoxideFormationbyNADH
withFlavins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
8.2.4 TheEffectofNicotinamideontheFADHydrolysis
andtheSuperoxideFormation. . . . . . . . . . . . . . . . . . . . 190
8.2.5 EffectofEDTAonFADHydrolysisandSuperoxide
Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
8.2.6 TheEffectofPyridineNucleotidesontheHydrolysis
ofFADandtheSuperoxideFormation. . . . . . . . . . . . . . 190
8.2.7 TheContributionofFMNtotheSuperoxide
Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
8.2.8 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
8.3 SomePropertiesofMitochondrialLipofuscin. . . . . . . . . . . . . . . 192
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Abbreviations
MC mitochondria
PMC proto-mitochondria
PostMC post-mitochondria
LF lipofuscin
NAD nicotinamideadeninedinucleotide
NADH nicotinamideadeninedinucleotidereduced
NADPH nicotinamideadeninedinucleotidephosphatereduced
FMN flavinmononucleotide
FAD flavinadeninedinucleotide
RF riboflavin
ATP adenosinetriphosphate
ADP adenosinediphosphate
AMP adenosinemonophosphate
EDTA ethylenediaminetetraaceticacid
SDS sodiumdodecylsulfate
RSandRI rotenone-sensitiveandrotenone-insensitive
DCPIP dichlorphenolindophenol
pNTV para-nitrotetrazoliumviolet
PA palmiticacid
DNA deoxyribonucleicacid
TPP tetraphenylphosphonium
TMR tetramethylrhodamine
UV ultraviolet
DTT dithiothreitol
PAAG polyacrylamidegel
MDA malonildialdehyde
TBA thiobarbituricacid
BSA bovineserumalbumin
xi
xii Abbreviations
ANS aniline-naphthalenesulfonate
M540 merocyanin-540
7AAMD 7-amino-actinomycinD
P polarizationdegree
τ lifetimeoftheexcitedstate