Energy-converting (NiFe) hydrogenases in archaea and bacteria [Elektronische Ressource] : insights into the energy-transducing mechanism / vorgelegt von Lucia Forzi

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Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Energy-converting [NiFe] hydrogenases
in archaea and bacteria:
insights into the energy-transducing mechanism







DISSERTATION


zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)








dem Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von




Lucia Forzi
aus Mailand/Italien



Marburg/Lahn im Juni 2005


Die Untersuchungen zur vorliegenden Arbeit wurden von März 2002 bis April 2005 am
Max-Planck-Institut für terrestrische Mikrobiologie unter der Leitung von Herrn PD
Dr. Reiner Hedderich durchgeführt.































Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation
angenommen am: 18.08.2005


Erstgutachter: PD Dr. Reiner Hedderich

Zweitgutachter: Prof. Dr. Wolfgang Buckel


Tag der mündlichen Prüfung am: 24.08.2005
Ein Teil der während der Promotion erzielten Ergebnisse wurde in folgender
Originalpublikation veröffentlicht:

Forzi, L., Koch, J., Guss, A. M., Radosevich, C. G., Metcalf, W. W. and Hedderich,
R. (2005). Assignment of the [4Fe-4S] clusters of Ech hydrogenase from
Methanosarcina barkeri to individual subunits via the characterisation of site-directed
mutants. FEBS Journal, 272, 4741-4753.

Die in dieser Arbeit erzielten Ergebnisse wurde im folgenden Übersichtsartikel dargestellt:

Hedderich, R. and Forzi, L.(2005). Energy-converting [NiFe] hydrogenases: more
than just H activation. Journal of Molecular Microbiology and Biotechnology, 2
eingereicht.













































Dedicated to my family



TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
1

I SUMMARY 2

I ZUSAMMENFASSUNG 4

II INTRODUCTION 7
1. Hydrogenases: general characteristics 7
2. Classes of hydrogenases 7
3. Structure of [NiFe] hydrogenases 8
4. Energy-converting [NiFe] hydrogenases 10
4.1 Ech hydrogenase from Methanosarcina barkeri 13
4.2 Related hydrogenases found in non-methanogenic microorganisms 16
5. Outline of this thesis 17

III MATERIALS AND METHODS 19
1. Materials used 19
1.1 Chemicals and biochemicals 19
1.2 Gases 19
1.3 Radioisotopes 19
1.4 Anaerobic buffers and solutions 20
1.5 Material used for protein purification 20
2. Microorganisms used and growth conditions 20
2.1 Microorganisms 20
2.2 Growth conditions 21
3. Biochemical methods 23
3.1 Preparation of cell extracts and isolation of membranes from M. barkeri
23
3.2 Purification of Ech hydrogenase from M. barkeri 23
3.2.1 Purificahy acetate grown M. barkeri strain
5 TABLE OF CONTENTS
Fusaro 23
3.2.2 Purification of Ech hydrogenase from methanol grown M. barkeri echF
mutant strains 24
3.3 Purification of ferredoxin from acetate grown M. barkeri 25
3.4 Determination of enzyme activities 25
3.5 Protein immunodetection by Western blot analysis 28
4. Analytical methods 29
4.1 Protein determination 29
4.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 29
4.3 Determination of ATP 30
4.4 Determination of H by gas chromatography 31 2
5. Measurements of ion translocation 31
5.1 Determination of proton translocation with a pH electrode 31
5.1.1 Proton translocation by suspensions of washed cells of
32 C. hydrogenoformans
5.2 Determination of sodium transport 32
6. Molecular biology methods 33
6.1 Construction of echF mutants 33
6.2 Plasmid and oligonucleotides 34
6.3 Isolation of plasmid DNA 35
6.4 Determination of DNA concentration 35
6.5 Digestion of plasmid DNA with restriction endonucleases 35
6.6 Agarose gel electrophoresis 36
6.7 Preparation and transformation of electrocompetent E. coli cells 36
6.8 Site-directed mutagenesis 37
6.9 DNA Sequencing 37
7. Biophysical methods 38
7.1 EPR spectroscopy studies 38
7.1.1 Introduction to EPR spectroscopy 38
7.1.2 Preparation of samples for EPR spectroscopy 40
7.1.3 EPR spectroscopy measurements 40
7.1.4 Evaluation of the spectra 41
7.1.5 Determination of the temperature-dependency of EPR signals 41
7.2 FT-IR spectroscopy 42
7.2.1 Introduction to FT-IR spectroscopy 42
6 TABLE OF CONTENTS
7.2.2 Electrochemistry 43
7.2.3 FT-IR spectroscopy measurements 43

IV RESULTS 45
1. Site-directed mutagenesis of conserved cysteine residues in subunit EchF
of Ech hydrogenase from Methanosarcina barkeri 45
1.1 Generation of echF mutants 45
1.2 Isolation of Ech hydrogenase from the echF mutant strains 49
1.3 EPR analysis of Ech hydrogenase isolated from echF mutant strains 51
2. Inhibitor studies with DCCD 57
2.1 Inhibition of Ech hydrogenase from M. barkeri by DCCD 58
2.2 Identification of Ech hydrogenase subunits modified by DCCD 60
3. FT-IR spectroscopic characterization of Ech hydrogenase from
Methanosarcina barkeri 63
3.1 Electrochemically induced FT-IR difference spectra 63
-1 3.2 Signals in the 2200–1800 cm range 63
-1 3.3 Signals in the 1800–1200 cm range 65
4. Identification of the coupling ion used by Coo hydrogenase from
68 Carboxydothermus hydrogenoformans
4.1 Determination of proton translocation with a pH electrode 68
22 + 4.2 Determination of sodium translocation with Na 70
4.3 Inactivation of Coo hydrogenase by DCCD in the presence of sodium ions
72

V DISCUSSION 74
1. Characterisation of the metal centers of Ech hydrogenase from
Methanosarcina barkeri by site-directed mutagenesis 74
1.1 Assignment of the [4Fe-4S] clusters of Ech hydrogenase to individual
subunits 74
1.2 Electron transfer pathway in Ech hydrogenase 78
1.3 Presence of an additional cofactor? 79
2. Role of the membrane part of energy-converting hydrogenases 80
80 2.1 Inhibitor studies with DCCD
2.2 Conserved acidic residues predicted to be located in transmembrane
82 helices
7 TABLE OF CONTENTS
3. FT-IR spectroscopic characterization of Ech hydrogenase from
88 Methanosarcina barkeri in comparison to complex I
4. Identification of the coupling ion used by Coo hydrogenase from
90 Carboxydothermus hydrogenoformans
95 5. A comparison of energy-converting hydrogenases and complex I

VI 99 REFERENCES

8 LIST OF ABBREVIATIONS

LIST OF ABBREVIATIONS

BV Benzyl viologen
CCCP Carbonylcyanide-m-chlorophenylhydrazone
CoM-S-S-CoB Heterodisulfide of H-S-CoM and H-S-CoB
DCCD N,N’-dicyclohexylcarbodiimide
DTT 1,4 Dithiothreitol
Standard Gibbs energy change at pH 7 ∆G°'
Transmembrane electrochemical proton gradient ∆µ + Η
+ Transmembrical sodium ion gradient ∆µ Na
Transmembrane gradient of protons ∆pH
Transmembrane electrical gradient (mV) ∆ Ψ
-1 -1Molar absorbtion coefficient (mM cm ) ε
E°' Standard redox potential at pH 7
Ech Energy converting hydrogenase
EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
EIPA 5-(N-ethyl-N-isopropyl)-amiloride
ETH-157 N,N’–dibenzyl-N,N’–diphenyl-1,2-phenylenedioxydiacetamide
Fd Ferredoxin
Hdr Heterodisulfide reductase
H-S-CoB 7-mercaptoheptanoylthreonine phosphate (coenzyme B)
H-S-CoM 2-mercaptoethanesulfonate (c