Molecular background of oxygen sensitivity in [FeFe] hydrogenases [Elektronische Ressource] / submitted by Sven T. Stripp

Strippsven

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• Biologie

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• Publié par: ruhr-universitat_bochum
• Publié le: 01 janvier 2010
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 6 Mo

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Molecular Background of Oxygen Sensitivity
in [FeFe] hydrogenases
Dissertation to obtain the degree
Doctor Rerum Naturalium (Dr. rer. Nat.)
at the faculty Biology and Biotechnology
Ruhr-University Bochum
International Graduate School of Biosciences
Ruhr-University Bochum
Department of Plant Biochemistry
Photobiotechnology
submitted by
Sven T. Stripp
Bochum
February 2010
I Molekulare Grundlagen
der Sauerstoffsensitivität
von [FeFe]-Hydrogenasen
Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
der Fakultät für Biologie und Biotechnologie
an der internationalen Graduiertenschule Biowissenschaften
der Ruhr-Universität Bochum
angefertigt im
Lehrstuhl Biochemie der Pflanzen
AG Photobiotechnologie
vorgelegt von
Sven T. Stripp
aus
Wesel
Bochum
Februar 2010
II Meinen geliebten Füchsen
What is happening to my skin?
Where is that protection that I needed?
Air can hurt you too
Air can hurt you too
Some people say not to worry about the air
Some people never had experience with...
Air...
Talking Heads
III TABLE OF CONTENTS
Table of figures
Introduction
1.1 Hydrogenases catalyse uptake and release of dihydrogen 1
Hydrogen in enlivened Nature 1
Convergent evolution gave three classes of hydrogenases 2
Structural differentiation in [FeFe] hydrogenases 5
The minimal [FeFe] hydrogenases in green algae 6
The C. reinhardtii hydrogenase serves as a role model
in [FeFe] hydrogenase research 7
1.2 Iron-sulphur proteins 8
Iron-sulphur clusters mediate single electron transport reactions 8
Iron-sulphur proteins are found in a variety of specialisations 8
Iron-sulphur proteins and the problem with oxygen 10
Results
Benchmark of contribution 13
2.1 Optimized over-expression of [FeFe] hydrogenases
with high specific activity in Clostridium acetobutylicum 14
2.2 Immobilization of the [FeFe] hydrogenase CrHydA1 on a gold electrode:
Design of a catalytic surface for the production of molecular hydrogen 21
2.3 The structure of the active site H-cluster of [FeFe] hydrogenase
from green algae Chlamydomonas reinhardtii studied
by X-ray absorption spectroscopy 29
2.4 How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms 38
IV 2.5 Electrochemical kinetic investigations of the reactions of [FeFe] hydrogenase
with CO and O : Comparing the importance of gas tunnels 2
and active-site electronic/ redox effects 45
2.6 How algae produce hydrogen –
News from the photosynthetic hydrogenase 57
Discussion
3.1 Heterologous expression and synthesis of [FeFe] hydrogenases 68
3.2 On the electronic structure of the H-cluster 70
3.3 Immobilisation of [FeFe] hydrogenases on conductive surfaces 71
Spectro-electrochemical analysis of hydrogenase films 72
Direct electrochemistry of hydrogenase films on graphite 72
3.4 Mechanisms of O inactivation in [FeFe] hydrogenases 73 2
[NiFe] and [FeFe] hydrogenases display different levels of O sensitivity 74 2
A model for the molecular mechanism of O inactivation 77 2
Towards the O -tolerant [FeFe] hydrogenase 80 2
Summary/ Zusammenfassung
Acknowledgments/ Danksagung
Literature
Curriculum vitae
Erklärung
V TABLE OF FIGURES
Figure 1 – The cofactor of [Fe] hydrogenases (Hmd)
Figure 2 – The Ni-Fe cofactor of [NiFe] hydrogenases
Figure 3 – The “H-cluster” of [FeFe] hydrogenases
Figure 4 – Different redox states of the H-cluster
Figure 5 – Iron-sulphur cluster arrangement in CpI
Figure 6 – Crystal structures of and DdH and CpI including [FeS] cluster equipment
Figure 7 – Homology model of the algal [FeFe] hydrogenase CrHydA1
Figure 8 – Iron-sulphur clusters in substrate binding and catalysis
Figure 9 – Position of the cofactors in [Fe] and [FeFe] hydrogenases relative to the protein surface
Figure 10 – Changes upon CO oxidation of the H-cluster as monitored by Fe–Fe distances
Figure 11 – Schematic comparison of the set-up in SEIRAS and graphite based electrochemistry
Figure 12 – Selective access to the active site in [FeFe] hydrogenase CpI
Figure 13 – Comparison of k for CO and O and the rate of reactivation after CO inhibition (k ) inact 2 re-act
Figure 14 – Stepwise degradation of the H-cluster by superoxide
VI INTRODUCTION
INTRODUCTION
1Hydrogen (H ) is the most abundant and lightweight element in the universe. The stable form of
hydrogen under standard conditions is “dihydrogen” H . Dihydrogen is an inert molecule with both 2
-1nuclei sharing two 1s electrons. The H molecule has a bond enthalpy of 436 kJ mol which reflects 2
its chemical robustness. This extraordinary high bonding energy makes H an excellent energy carrier. 2
Its specific enthalpy is four-fold that of coal, and about 2.5-fold that of diesel or natural gas [1]. With
just two electrons shared, the intermolecular attraction of nuclei is weak. Thus, H is highly volatile 2
and only trace levels are left in the lower atmosphere [2]. Dihydrogen escaped the stratosphere due to
either diffusion [3] or oxidation in the course of the onset of oxygenic photosynthesis [4, 5]. Hydrogen
- +is scarcely found in the H form but as hydride ion (H ) and proton cation (H ) in water, Earth’s crust, 2
and all kinds of life.
1.1 Hydrogenases catalyse uptake and release of dihydrogen
Hydrogen in enlivened Nature
In anaerobic segments of deep lakes and hot springs the concentration of H is much higher than in the 2
stratosphere. Strict anaerobe bacteria and archaea make use of protons as terminal electron acceptor in
anaerobic respiration and fermentation. Oxidation of organic matter and generation of ATP is coupled
to reduction of protons that alternatively stand in for O . Anaerobic respiration is not the only 2
metabolic pathway that produces H . In carboxytrophic bacteria of the Carboxydothermus genus [6] 2
oxidation of CO to CO is often coupled to proton reduction [7]. Nitrogen–fixing archaea, 2
proteobacteria (e.g., root nodule bacteria Rhizobia spec.), and cyanobacteria (Nostoc, Anabaena)
release H as a by-product in the reduction of inorganic N to bio-available NH [8-10]. Nitrogen-2 2 3
fixation is catalysed by the nitrogenase complex and demands an additional energy input of
16 equivalents ATP per N and H . 2 2
Microbial H release is versatile and typically occurs under anaerobic conditions. However, 2
H uptake is found in many micro organisms as well. Knallgas bacteria and affiliated species of the 2
Desulvovibrio genus use H as a source of electrons to power their metabolism [11, 12]. Interestingly, 2
the notoriety for anaerobiosis is much less pronounced in organisms relying on H uptake than it is 2
found with H release [13]. The non-standard name “Knallgas bacteria” refers to the fact that bacteria 2
like Ralstonia eutropha and Hydrogenobacter spec. can live lithoautotrophically on a mixture of
H and O [14]. 2 2
1 INTRODUCTION
Convergent evolution gave three classes of hydrogenases
Hydrogenases are oxido-reductases (EC 1.12) that catalyse uptake and evolution of H with a variety 2
+ –of redox partners. The reaction hydrogenases perform reads 2 H + 2 e H . While the nitrogenase-2
based hydrogen metabolism is a physiological specialisation, hydrogenases are ubiquitous in strict and
facultative anaerobes, including some unicellular eukaryotes [15]. Three classes evolved
independently: [NiFe], [FeFe] and [Fe] hydrogenases. These types of hydrogenase enzymes do not
share common ancestors [16]. The mechanistic similarities, however, are striking and although this
work will focus on [FeFe] hydrogenases it is worth learning about hydrogen catalysis in [Fe] and
[NiFe] hydrogenases as well.
[Fe] hydrogenases (Hmd) (also known as “[FeS] cluster-free hydrogenases”) have been described for
a number of methanogenic archaea. Originally discovered in Methanothermobacter marburgensis
[17], the hydrogenase of Methanocaldococcus jannaschii has been crystallized just recently [18, 19].
[Fe] hydrogenases catalyse the uptake of H in a binary reaction. 2
[Fe] hydrogenases incorporate a
(A)
O N
Nlow-spin iron atom bound to a O(B) 1O N(C) cysteine residue. Two intrinsic CO P
S
N
NOligands coordinate this central metal
N
O
Xion [20]. A 2-pyridinol compound O
O
O
P O(“FeGP”) binds the iron atom at two O
O O
sites presumably: via pyridinol-N and
Figure 1 – The cofactor of [Fe] hydrogenases (Hmd). The central a formyl carbon side chain [21].
iron atom (red, A) is coordinated by a single cysteine thiolate and
Figure 1 shows a schematic drawing holds two CO groups. The nitrogen atom of the pyridine ring and
a formyl carbon atom (1) bind to the iron compound as well. Due
of the [Fe] hydrogenase cofactor
to its octahedral geometry, one binding site is vacant (X). The
FeGP cofactor is a carboxymethyl-3,5-dimethyl-2-pyridone-4-yl arrangement. Due to its octahedral
(B) bound to 5’-guanosyl (C) via a phosphate group. The non-iron
+ geometry, the central iron atom has a cofactor methenyl-H MPT is not shown (see text).4
vacant binding site. Dihydrogen is
+ –dissociated heterolytical