Probing the Dynamical Basis of Radical Catalysis in a B12 Dependent Enzyme and Development of a Cobalamin/Protein Compressed Photosynthesis Device Open Access
Robertson, Wesley Daniel (2010)
Abstract
The contribution of chemical and protein coordinates, and the
influence of substrate
binding, to the formation and stabilization of the
cob(II)alamin-5'-deoxyadenosyl radical
pair were studied in the adenosylcobalamin (AdoCbl)-dependent
enzyme, ethanolamine
ammonia-lyase (EAL) from Salmonella typhimurium. The
photoproduct dynamics on the
10-7 - 10-1 s time scale were monitored following pulsed-laser
photolysis with a home
designed and constructed ultraviolet(UV)/visible transient
absorption spectrometer.
Pulsed-laser photolysis of AdoCbl in EAL leads to a quantum yield
for
cob(II)alamin which is 3-fold less than for AdoCbl in aqueous
solution at 295 K,
indicating that the protein binding site suppresses photoproduct
radical pair formation.
The quantum yield was further reduced by half following the binding
of the substrate
analog, ( S)-1-amino-2-propanol, suggesting that it does not
induce changes in the protein
that are characteristic of true substrates. Therefore, a
catalytically competent ternary
complex, with negligible turnover on the photolysis/probe time
scale, was created by
using a fluid dimethylsulfoxide/water cryosolvent system at 230 -
240 K. The static
UV/visible absorption spectra of holo-EAL and ternary complex are
comparable,
indicating that the binding of substrate does not weaken the
cofactor cobalt-carbon (Co-
C) bond by significantly distorting AdoCbl structure. The results
indicate that substrate binding
to holo-EAL does not "switch" the protein to a new static
structural state, which
stabilizes the cob(II)alamin-5'-deoxyadenosyl radical pair
photoproduct. Therefore,
protein dynamics plays a critical role in native cleavage of the
Co-C bond and radical pair
separation. The EutB subunit of the EAL enzyme was selected as a
template for the
design of an artificial photosynthetic unit for the reduction of
carbon dioxide (CO2) and
toxic halo-organic compounds. Cobalamin (Cbl) or cobinamide (Cbi)
in solution and
bound to EutB were chemically or photo reduced to the CoI state.
Interaction of the
compounds with CO2 and halo organics was monitored by using
UV/visible
spectroscopy. The results suggest CO2 binding to the CoI centers,
and light-driven
haloalkane reduction.
Table of Contents
Chapter 1: Introduction 1.1 The Coenzyme B12-Dependent Enzyme Ethanolomine Ammonia Lyase 1.2 Proposed Mechanisms for Cobalt-Carbon Bound Cleavage Enhancement Chemomechanical Strain Hypothesis Dynamic Contributions to Catalysis 1.3 Photolysis of Cobalamins 1.4 Outline of Dissertation Chapter 2: Construction of a UV/Visible Transient Absorption Spectrometer 2.1 Transient Absorption Spectroscopy: Principles 2.2 Mechanical and Optical Setup Transient Absorption Spectrometer Housing and Sample Holder Sample Excitation Probe Beam Configuration Probe Beam Detection 2.3 Instrument Control and Data Acquisition Timing and Interface Chapter 3: Static and Transient Absorption Studies of Adenosylcobalamin and Methylcobalamin in Ethanolamine Ammonia-Lyase: Influence of Substrate/Inhibitor Binding on Cofactor Spectra and Radical Pair Recombination Synopsis 3.1 Materials and Methods Sample Preparation Static Absorption Spectroscopy Transient Absorption Spectroscopy 3.2 UV/Visible Absorption Spectra of Cobalamins Following Binding to EAL with and without Substrate and Substrate Analogs Pre-Photolysis Absorption Spectra of Adenosylcobalamin and Methylcobalamin Complexes Post-Photolysis Absorption Spectra of Adenosylcobalamin and Methylcobalamin Complexes 3.3 Quantum yield of cob(II)alamin formation following photolysis of AdoCbl and MeCbl in solution and in EAL Quantum Yield Measurements of Adenosylcobalamin in EAL with Substrate Inhibitor Quantum Yield Measurements of Methylcobalamin in EAL with Native Substrates and Substrate Inhibitors 3.4 Time-dependence of photoproduct cob(II)alamin following photolysis of AdoCbl and MeCbl in solution and in EAL Adenosylcobalamin Methylcobalamin 3.5 Discussion Model for the Formation and Reaction of Cage Escaped CoII-Radical Pair Populations Following AdoCbl and MeCbl Photolysis in EAL Substrate Analog Binding Influences the Cage Escape Process Estimation of Cage Escape Rate Constants and Activation Free Energy Barriers Implications for the Substrate Trigger Mechanism of Cob(II)alamin-5'-Deoxyadenosyl Radical Pair Formation in EAL Chapter 4: Characterization of Contributions of Protein and Chemical Coordinates to the Substrate Trigger of Cobalt-Carbon Bond Cleavage and Radical Pair Separation in the Ternary Complex of Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase Synopsis 4.1 Materials and Methods Materials Enzyme Sample Preparation Adenosylcobalamin Sample Preparation Low Temperature Optical Cryostat Low Temperature Static Absorption Spectra Low Temperature Transient Absorption Spectroscopy EPR Spectroscopy of Cob(II)alamin-Substrate Radical Pair Formation Quantum Yield Measurements Temperature-Dependence of the First-Order Rate Constant 4.2 UV/visible Absorption Spectra of Holoenzyme and Ternary Complex at 230 K 4.3 UV/visible Transient Absorption Spectroscopy of Holoenzyme and Ternary Complex at 230 K Quantum Yield of Cob(II)alamin Formation following Low Temperature Photolysis of AdoCbl in Solution, Holoenzyme and Ternary Complex Time Dependence of Photoproduct Cob(II)alamin Recombination Following Photolysis Under Saturating Pulsed-Laser Irradiation Visible Light Irradiation of the EAL Ternary Complex Does Not Generate the Cob(II)alamin-Substrate Radical Pair Effect of Photolysis on Thermally-Activated Cob(II)alamin-Substrate Radical Pair Formation 4.4 Discussion Substrate Binding to Holo-EAL Does Not Activate the Co-C Bond for Cleavage by Distorting AdoCbl Structure Substrate Binding to Holo-EAL Does Not Elicit Prompt Stabilization of the Cob(II)alamin-Radical Pair Photoproduct Substrate Binding to Holo-EAL Does Not Significantly Influence Cage Escape of the Radical Pair Substrate Binding to Holo-EAL Does Not Significantly Alter Stabilization of the Cage-Escaped Cob(II)alamin-Radical Pair Photoproduct Models for the Substrate-Initiated Co-C Bond Activation and Cleavage 4.5 Conclusions Chapter 5: Developing a Biologically-Inspired Molecular Solar Energy Conversion Device: Reaction of Solution and Protein-Bound Cobalamin Cofactors with Carbon Dioxide and Halo-Organic Compounds Synopsis 5.1 Introduction Compressed Photosynthesis Construct Reactions of Metal Complexes with CO2 and Halo-Organics 5.2 Materials and Methods Sample Preparation Preparation of pH controlled MeCbi Samples Anaerobic Techniques Titanium(III) Citrate Preparation Nuclear Magnetic Resonance (NMR) Product Analysis UV/Visible Absorption Spectroscopy Synthesis of Methylcobinamide (MeCbi) 5.3 Reduction of Cobalamin and Cobinamide Reduction of Cobalamin Cobinamide Comparison of Cobalamin and Cobinamide Reduction Conclusion 5.4 Oxidation of Cob(I)alamin and Cob(I)inamide in the Presence of CO2. Reaction of Cob(I)inamide in Aqueous Solution at pH 6.0 Reaction of cob(I)inamide with CO2 in aqueous solution at pH 6.0 5.5 Reaction of Cobinamide with Chloroacetonitrile in Aqueous Solution at pH 7.5 5.6 Discussion Dihydrogen formation CO2 Binding Interaction with Cob(I)inamide 5.7 Reduction of Cobalamin and Cobinamide Cofactors Bound to EAL and EutB Protein Reduction of Cobalamin Bound to EAL and EutB Reduction of Cobinamide Bound to EAL and EutB Conclusion Appendix Bibliography
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