Optimizing Photo-reduction Methods for the Light Induced Turnover of [FeFe] Hydrogenase Open Access
Sanchez, Monica (Fall 2020)
Abstract
Hydrogenases are a class of oxidoreductase enzyme which catalyze the reduction of protons to molecules of hydrogen, as well as the oxidation of hydrogen, at the fastest rates ever recorded. They do so with almost no over potential, while using earth abundant transition metals. To reproduce their unique strengths as catalysts, a detailed understanding of their chemical mechanism is required. However, it is precisely their own efficiency that presents barriers to study and replication. Due to their exceedingly rapid turnover rate, researchers have consistently struggled to map their mechanism in detail, and the research that has been produced has been characterized by conflicting data, and thus a lack of cohesive interpretation. In this thesis, a method for rapidly inducing turnover is explored, optimized and finally employed for studying two different [FeFe] hydrogenases, one from Chlamydomonas reinhardtii (CrHydA1) and the other from Desulfovibrio desulfuricans (DdHydAB).
Steady state and transient kinetic analysis of viologen based redox mediators was used to probe the ET mechanism between a nanocrystalline semiconductor and a NiFe hydrogenase. The results from this study revealed a complex interplay between the structure and the LUMO energy of the mediator, which could be tuned to optimize ET to hydrogenase. Despite significant advancement in the efficiency of the technique gained through this study, optimization of the for application to transient studies under varying experimental conditions remained to be explored. The results of this subsequent study led to the outlining of principles for achieving a specific solution potential based on the tuning of sample conditions.
Finally, once the details for this photo-initiation technique were outlined, the guidelines established by the previous studies were applied to the mechanistic study of two [FeFe] hydrogenases: CrHydA1 and DdHydAB. Both enzymes were investigated through time resolved spectroscopy, revealing rich intermediate dynamics on a previously unexplored timescale. The active site dynamics probed by time resolved infrared transient absorbance demonstrated the kinetic competency of several intermediate states previously proposed. Furthermore, preliminary results on the mechanism of the chemical reactions necessary for inter-conversion of the verified intermediates are reported, and suggest the likely pathways involved in enzymatic turnover.
Table of Contents
Chapter 1: Introduction
1.1 – The Global Energy Crisis and the Storage of Energy in Small Molecule Bonds
2
1.2 - Efficient Oxidoreductase Enzymes for Small Molecule Synthesis and Solar Fuels
6
1.3 – Hydrogenases: Structure, Efficiency and Challenges to Study
9
1.4 – Overview of [FeFe] Hydrogenases Catalytic Mechanism and Proposed Intermediates
11
1.5 – The “Potential Jump” Method for sub-Turnover Kinetics Analysis
14
1.6 – Hypothesis and Scope of Thesis
17
1.7 – References
20
Chapter 2: Materials and Methods
2.1 - Introduction
31
2.2 - Mediators Synthesis
32
2.2.1 - Preparation of 2-carbon Linker Mediators: DQ52 and DQ42
33
2.2.2 - Preparation of 3-carbon Linker Mediators: DQ03, DQ53 and DQ43
34
2.3 – Synthesis and Manipulation of Semi-Conducting Nanomaterials: CdSe QDs, CdSe/CdS Dot-in-rod and CdSe Nanorods
34
2.3.1 - Preparation of CdSe Quantum Dots
35
2.3.2 - Preparation of CdSe/CdS Dot-in-Rod (DIR) Nanorods
35
2.3.3 - Preparation of CdSe Nanorods
36
2.3.4 - Ligand Exchange of Nanoparticles for use in Aqueous Media
37
2.4.1 Buffers
37
2.5 - Analytical Methods
38
2.5.1 - UV/Vis Absorbance
38
2.5.2 – Photoluminescence Spectra
38
2.5.3 – Fourier Transform Infrared Spectroscopy (FTIR)
39
2.5.4 – Transmission Electron Microscopy (TEM)
40
2.5.5 – Time Correlated Single Photon Counting (TCSPC)
40
2.5.6 – In tandem Transient IR and Visible Absorbance
41
2.5.7 – Ultrafast Transient Visible Absorbance
42
2.5.8 – Nanosecond Transient Visible Absorbance Spectroscopy
43
2.5.9 – Electrochemistry: Cyclic Voltammetry
43
2.5.10 – Electrochemistry: Chronopotentiometry
44
2.5.11 – H NMR Spectroscopy
45
2.5.12 – X-ray Quality Crystal Growth Technique
45
2.6 – Light Driven Activity Assays and Quantum Yield Calculation
46
2.6.1 – Light-driven Mediator Reduction
46
2.6.2 – Light-driven H2 Production
47
2.6.3 – H2 Detection via a Pressure Sensor
48
2.7 – Data Analysis
49
2.7.1 – Fitting Infrared Transient Absorbance Data
49
2.7.2 – Fitting Visible Transient Absorbance Data
51
2.7.3 – Global Fitting FTIR and Absorbance Spectra
51
2.8 – References
52
Chapter 3 Optimizing Electron Transfer from CdSe QDs to Hydrogenase for Photocatalytic H2 Production
3.1 – Abstract
55
3.2 – Introduction
56
3.3 – Results and Discussion
57
3.3.1 – Characterization and Analysis of Mediator Structural and Electronic Properties by UV-Vis, H NMR, X-ray Crystallography, and Cyclic Voltammetry
57
3.3.2 – Determining the Extinction Coefficient for each 1-electron Reduced Mediator via Chronopotentiometry
61
3.3.3 – Quantum Efficiency of Photo-driven Mediator Reduction
64
3.3.4 – Quantum Efficiency of Photo-driven Hydrogen Production
67
3.3.5 – Spectral Characteristics of Mediator Radical Decomposition Product
72
3.4 – Transient Studies of Electron Quenching by Mediator Population and Charge Recombination
75
3.4.1 – Excited State Electron Quenching by Mediator LUMO
75
3.4.2 – Mediator Radical Charge Recombination
77
3.5 - Conclusions
80
3.6 – Appendix with Supplementary Data
81
3.6.1 H NMR spectrum for each mediator
82-86
3.6.2 Cyclic Voltammograms for each mediator
87
3.6.3 Summary of X-ray Diffraction Data
89
3.7 - References
90
Chapter 4 “The Laser-Induced Potential Jump: a Method for Rapid Electron Injection into Oxidoreductase Enzymes”
4.1 – Abstract
97
4.2 – Introduction
98
4.3 – Results and Discussion
101
4.3.1 – System Components: Photosensitizer, Mediators and Catalyst
101
4.3.2 – Steady-state Quantum Yield of Photo-driven Mediator Reduction and H2 Production
103
4.3.3 – Factors Affecting Potential Jump
109
4.3.4 – Kinetics of ET to CrHydA1 Hydrogenase
115
4.4 – Utilization of CdSe NRs for 532 nm Excitation
119
4.4.1 – Steady State Photo-driven Mediator Reduction
121
4.4.2 – Transient Potential Pump-Power Dependence with CdSe NRs
123
4.5 – Conclusions
124
4.6 – References
128
Chapter 5 Mechanistic Studies of [FeFe] Hydrogenase: CrHydA1 and DdHydAB
5.1 – Abstract
137
5.2 – Introduction
138
5.3 – Investigating the Kinetic Competency of Proposed Intermediates
141
5.3.1 – Rapid Initiation of Hydrogenase Turnover using a Potential Jump
141
5.3.2 – Photoprotection of the Enzyme by Photosensitizer
144
5.3.3 – H-cluster Sub-turnover Kinetics at pH 8.4
146
5.3.4 – TRIR Spectra Reveal a New Intermediate State
152
5.3.5 – Conclusions on the Kinetic Competency of Proposed Intermediate States
154
5.4 – CrHydA1 Mechanistic Studies with PDT mutant
157
5.4.1 – Light Titration Experiments with the PDT Mutant
159
5.4.2 – Kinetics Study of PDT Mutant under pHs Ranging from pH 6.5 to pH 10
162
5.5 – DdHydAB Mechanistic Studies
166
5.5.1 - pH Dependence of Intermediate State Transitions
167
5.2.2 – Redox Anti-cooperativity with PDT mutant
173
5.6 – Conclusions on [FeFe] Hydrogenase Mechanistic Studies
180
5.7 – References
181
Chapter 6: Conclusions and Future Directions
6.1 – Conclusions from
189
4.2 – Introduction
190
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