Optimizing Photo-reduction Methods for the Light Induced Turnover of [FeFe] Hydrogenase Pubblico

Sanchez, Monica (Fall 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/w3763802c?locale=it
Published

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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