Time-resolved surface enhanced resonance Raman spectro-electrochemistry of heme proteins [Elektronische Ressource] / Marc Groerüschkamp

Sujets

• Naturwissenschaften

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 4
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

Time-Resolved Surface
Enhanced Resonance Raman
Spectro-Electrochemistry of
Heme Proteins
D I S S E R T A T I O N
zur Erlangung des Grades
"Doktor der Naturwissenschaften"
eingereicht
am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg-Universit at in Mainz
Marc Gro erusc hkamp
geboren in Frankfurt am Main
Mainz, 08. November 2010II
|||||||||||||||||||||{
Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mundlic hen Prufung: 09.12.2010III
Die vorliegende Arbeit wurde am Max-Planck-Institut fur Polymerforschung
in Mainz und am Austrian Institute of Technology (AIT) in Wien in der Zeit
von Januar 2008 bis Dezember 2010 angefertigt.IVV
Abstract
The membrane protein Cytochrome c Oxidase (CcO) is one of the most im-
portant functional bio-molecules. It appears in almost every eukaryotic cell
and many bacteria. Although the di erent species di er in the number of
subunits, the functional di erences are merely marginal. CcO is the termi-
nal link in the electron transfer pathway of the mitochondrial respiratory
chain. Electrons transferred to the catalytic center of the enzyme conduce
to the reduction of molecular oxygen to water. Oxygen reduction is coupled
to the pumping of protons into the inter-membrane space and hence gen-
erates a di erence in electrochemical potential of protons across the inner
mitochondrial membrane. This potential di erence drives the synthesis of
adenosine triphosphate (ATP), which is the universal energy carrier within
all biological cells. The goal of the present work is to contribute to a better
understanding of the functional mechanisms of CcO by using time-resolved
surface enhanced resonance Raman spectroscopy (TR-SERRS). Despite in-
tensive research e ort within the last decades, the functional mechanism of
CcO is still subject to controversial discussions.
It was the primary goal of this dissertation to initiate electron transfer to
the redox centers Cu , heme a, heme a and Cu electrochemically and toA 3 B
observe the corresponding redox transitions in-situ with a focus on the two
heme structures by using SERRS. A measuring cell was developed, which
allowed combination of electrochemical excitation with Raman spectroscopy
for the purpose of performing the accordant measurements. Cytochrome c
was used as a benchmark system to test the new measuring cell and to prove
the feasibility of appropriate Raman measurements. In contrast to CcO
the heme protein cc contains only a single heme structure. Nevertheless,
characteristic Raman bands of the hemes can be observed for both proteins.
In order to investigate CcO it was immobilized on top of a silver sub-
strate and embedded into an arti cial membrane. The catalytic activity of
CcO and therefore the complete functional capability of the enzyme withinVI
the biomimetic membrane architecture was veri ed using cyclic voltamme-
try. Raman spectroscopy was performed using a special nano-structured sil-
ver surface, which was developed within the scope of the present work. This
new substrate combined two fundamental properties. It facilitated the for-
mation of a protein tethered bilayer lipid membrane (ptBLM) and it allowed
obtaining Raman spectra with su cient high signal-to-noise ratios.
Spectro-electrochemical investigations showed that at open circuit po-
tential the enzyme exists in a mixed-valence state, with heme a and and
heme a in the reduced and oxidized state, respectively. This was considered3
as an intermediate state between the non-activated and the fully activated
state of CcO. Time-resolved SERRS measurements revealed that a hampered
electron transfer to the redox center heme a characterizes this intermediate3
state.Contents
1 Motivation 1
2 Introduction 5
2.1 The Mitochondrial Respiratory Chain . . . . . . . . . . . . . . 5
2.1.1 Cytochrome c Oxidase . . . . . . . . . . . . . . . . . . 8
2.1.2 Cytochrome c . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Membrane Proteins and Biomimetic Architectures . . . . . . . 9
2.2.1 Protein Tethered Bilayer Lipid Membrane . . . . . . . 12
3 Theory 17
3.1 Raman Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.1 Molecular Vibrations . . . . . . . . . . . . . . . . . . . 17
3.1.2 Raman E ect . . . . . . . . . . . . . . . . . . . . . . . 17
3.1.3 Resonance E ect . . . . . . . . . . . . . . . . . . . . . 22
3.1.4 Surface Enhancement . . . . . . . . . . . . . . . . . . . 25
3.2 Surface Plasmon Resonance Spectroscopy . . . . . . . . . . . . 36
3.2.1 Surface Plasmon Excitation . . . . . . . . . . . . . . . 37
3.2.2 Detecting Adsorption by SPRS . . . . . . . . . . . . . 38
3.3 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3.1 Electrochemical Impedance Spectroscopy (EIS) . . . . 42
3.3.2 Cyclic Voltammetry (CV) . . . . . . . . . . . . . . . . 44
4 Materials and Methods 47
4.1 Sample Preparation Procedures . . . . . . . . . . . . . . . . . 47
4.1.1 Template Stripped Gold - TSG . . . . . . . . . . . . . 47
VIIVIII CONTENTS
4.1.2 Template Stripped Silver - TSS . . . . . . . . . . . . . 49
4.1.3 SERRS Substrates . . . . . . . . . . . . . . . . . . . . 49
4.1.4 Preparation of Cytochrome c Samples for SERRS . . . 51
4.1.5 Protein Tethered Bilayer Lipid Membrane (ptBLM) . . 51
4.2 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3 Surface Plasmon Resonance Spectroscopy . . . . . . . . . . . . 52
4.3.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.4 Spectroelectrochemistry . . . . . . . . . . . . . . . . . . . . . 55
4.4.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.4.2 Time-Resolved Measurements . . . . . . . . . . . . . . 57
4.5 Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5.1 Dynamic Light Scattering . . . . . . . . . . . . . . . . 58
4.5.2 Scanning Electron Microscopy . . . . . . . . . . . . . . 59
4.5.3 Atomic Force Microscopy . . . . . . . . . . . . . . . . . 59
5 Results and Discussion 61
5.1 A Novel Measuring Cell Design for SERRS Applications . . . 61
5.1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 61
5.1.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.1.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.2 Electron Transfer Kinetics of Cytochrome c Probed by Time-
Resolved SERRS . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 66
5.2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.2.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 74
5.3 Silver Surfaces with Optimized Surface Enhancement by Self-
Assembly of Silver Nanoparticles for Spectroelectrochemical
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 75
5.3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
5.3.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 91CONTENTS IX
5.4 Cytochome c Oxidase: Electrochemically Induced Electron
Transfer Probed by Surface Enhanced Resonance Raman Spec-
troscopy (SERRS) . . . . . . . . . . . . . . . . . . . . . . . . 92
5.4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 92
5.4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5.4.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.5 Electron Transfer Kinetics of Cytochrome c Oxidase Probed
by Time-Resolved Surface Enhanced Resonance Raman Spec-
troscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 108
5.5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.5.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 119
6 Final Conclusion 125
Bibliography 135
Appendix 156X CONTENTS