The Bioinorganic Energy Conversion Puzzle: Pieces from a [NiFe]-Hydrogenase Ligand Scaffold Open Access

Abstract

The activation and formation of small molecules by metalloenzymes is a critical component of microbial metabolism. It is also relevant to fuel production. Sustainable energy conversion catalysts are often modelled off such enzymes. However, they typically fall short of achieving the rates and/or efficiencies of the enzymes. Advances in (bio)inorganic catalysis have demonstrated the importance of the ligand/protein scaffold for optimal enzyme/catalyst function. Thus, understanding the roles of protein scaffolds in metalloenzymes is critical for refining bioinspired catalyst blueprints. In this thesis, the structure-function relationship of the [NiFe] active site with part of its secondary sphere is probed in the soluble hydrogenase-1 from Pyrococcus furiousus (Pf SH1) with three enzyme variants that targeted two critical amino acid residues.

The first two enzyme variants targeted a glutamate residue (E17) and changed either side chain functionality or length. The rather conservative mutation E17Q changed side chain functionality. Probing the effects of the mutation provided insight into/verification of a probable efficient proton transfer pathway. Subtle modulation of active site properties under equilibrium and time-resolved conditions were also observed. The less conservative mutation E17D changed the side chain length. Probing the effects of this mutation provided insight into the convoluted role of the [NiFe]-core with the secondary and outer coordination spheres. The third variant targeted an arginine residue above the active site exogenous ligand binding position (R355), which was mutated to a lysine (R355K). This thesis presents the first in depth spectroscopic characterization of the effects caused by this mutation to help determine possible role(s) of R355 during catalysis. In combination with H/D exchange kinetics, it was found a major role of the arginine is to control the reactivity and/or stability of bridging hydrides that are critical catalytic intermediates.

Overall, the results established fundamental secondary sphere interactions with the [NiFe] core in Pf SH1, which sets a foundation for more in depth equilibrium, steady state, and time-resolved investigations of Pf SH1 enzyme variants.

Table of Contents

Chapter 1 – Introduction ... 1

1.1 – Overview of Pollution, Global Warming, the Need for Renewable Energy, and Renewable Energy Inspirations found in Nature ... 2

1.1.1 – Motivation: Fossil Fuel Contributions to Pollution and Climate Change and the Need for Alternative Energy Sources ... 2

1.1.2 – A Brief Overview of Promises and Inherent Challenges in Nuclear and Renewable Energy and the Need for Efficient Alternative Fuel Generation ... 3

1.2 – Enzymatic Catalysis and Gas Processing Metalloenzymes for Energy Conversion: General Considerations ... 10

1.3 – Hydrogenases, with a Focus on [NiFe]-Hydrogenases ... 13

1.3.1 – An Overview of Hydrogenases and Their General Reactivity ... 13

1.3.2 – [NiFe]-Hydrogenases: General Properties ... 16

1.3.3 – [NiFe]-Hydrogenases: A Mechanistic Outline in the Primary Sphere ... 20

1.3.4 – A Need to Probe Structure-Function Relationships Between the Active Site and Ligand Scaffold ... 24

1.4 – Hypothesis and Scope of this Thesis ... 29

1.5 – References ... 29

Chapter 2 – Materials, Methods, and Data Analysis ... 47

2.1 – Introduction ... 48

2.2 – Preparation of Native Soluble Hydrogenase – 1 and the E17Q, E17D, and R355K variants ... 48

2.3 – Materials ... 49

2.3.1 – Generals Considerations ... 49

2.3.2 – Buffers ... 50

2.3.3 – Synthesis of 1,1’-trimethylene-2,2’-bipyridine dibromide ... 50

2.3.4 – CdSe/CdS Dot-in-Rod Nanocrystal Synthesis, Ligand Exchange, and Purification ... 52

2.3.5 – CdS Rod Nanocrystal Synthesis, Ligand Exchange, and Purification ... 52

2.4 – Methodology ... 55

2.4.1 – Standard Hydrogen Oxidation and Proton Reduction Activity ... 55

2.4.2 – Standard UV-Vis Spectroscopy ... 56

2.4.3 – Electron Paramagnetic Resonance Spectroscopy of Native and R355K Pf SH1 ... 56

2.4.4 – Preparation of Samples for Infrared Spectroscopy ... 57

2.4.4.1: Native, E17D, and R355K Pf SH1 Photochemical Reduction Samples ... 57

2.4.4.2: Native, E17Q, and R355K Pf SH1 CO Incubated Samples ... 58

2.4.4.3: E17Q Pf SH1 pH Dependent Samples ... 59

2.4.4.4: E17D Pf SH1 pH Dependent Samples ... 59

2.4.4.5: R355K Pf SH1 pH Dependent Samples ... 60      

2.4.5 – Equilibrium Photochemical Reduction of PDQ²⁺ with CdSe NCs and Quantum Efficiency ... 60

2.4.6 – Fourier Transform Infrared Spectroscopy Measurements, Including Temperature Dependent Measurements ... 61

2.4.7 – Equilibrium Photochemical Reduction Coupled to Fourier Transform Infrared Spectroscopy with Native, E17D, and R355K Pf SH1 ... 62

2.4.8 – Time Resolved Infrared Spectroscopy ... 63

2.4.9 – H/D Exchange with Native and R355K Pf SH1 ... 65

2.4.10 – Methyl Viologen Reduction by Native SH1 and R355K Pf SH1 under 5% H2 Monitored via UV-Vis Spectroscopy ... 66

2.4.11 – Sequence Alignments ... 66

2.5 – Data Analysis ... 67

2.5.1 – Quantum Efficiency of Photochemical Reduction of PDQ²⁺ with CdSe NCs ... 67

2.5.2 – EPR spectroscopy ... 69

2.5.3 – Fourier Transform Infrared Spectroscopy: Equilibrium Photochemical Reduction of Native, E17D, and R355K SH1 ... 69

2.5.4 – Standard Fourier Transform Infrared Spectroscopy ... 70

2.5.5 – Transient Infrared Absorption of CO Photolysis and Hydride Photolysis ... 71

2.5.6 – IR Spectroscopy Analysis Specific to R355K Pf SH1 ... 73

2.5.7 – H/D Exchange ... 75

2.6 – References ... 76

Chapter 3 – Native Pf SH1 and Investigating the E17Q and E17D Variants ... 80

3.1 – Introduction ... 81

3.2 – Results and Discussion ... 85

3.2.1 – Native Pf SH1 Equilibrium Photochemical Reduction, Electron Paramagnetic Resonance, and Time Resolved Exogenous Carbon Monoxide and Hydride Photolysis ... 85

3.2.2 – Investigations of E17Q Pf SH1: Equilibrium FTIR, Time Resolved Hydride Photolysis, and Exogenous CO Photolysis ... 100

3.2.3 – Investigations of E17D Pf SH1: Equilibrium FTIR and FTIR Photochemical Reduction ... 113

3.3 – Conclusions ... 119

3.4 – References ... 121

Chapter 4 – Investigating the R355K Variant ... 133

4.1 – Introduction ... 134

4.2 – Results ... 140

4.2.1 – Steady State Kinetics ... 140

4.2.2 – Electron Paramagnetic Resonance Spectroscopy ... 140

4.2.3 – Fourier Transform Infrared Spectroscopy ... 143

4.3 – Discussion ... 150

4.4 – Conclusions ... 156

4. 5 – References ... 156

Appendix ... 167

Appendix A – Additional Electron Paramagnetic Resonance Spectroscopy Plots and Tabulated g-values ... 168

Appendix B – Native Pf SH1 FTIR Auto-Oxidation Monitored by FTIR and Additional E17Q Auto-Oxidation FTIR Plots ... 174

Appendix C – Second Derivative Spectra, Non-Normalized E17D pH Dependent Spectra, and the CN Region from the Photochemical Reduction ... 176

Appendix D – H/D Exchange Calibration, Data Analysis and, and Additional Analysis ... 178

Appendix E – Second Derivative Spectra, Absorbance Spectra, and the νCN Region Difference Spectra from R355K Equilibrium Photochemical Reduction ... 182

Appendix F – Second Derivative Spectra, Non-normalized Absorbance Spectra, Normalization Values, Peak Positions and Peak Areas Before and After Oscillator Strength Correction of the pH Dependent R355K FTIR Spectra ... 184

Appendix G – Methyl Viologen Reduction Under 5% H2 Monitored by UV-Vis Spectroscopy ... 187

Appendix H – Second Derivatives of the R355K pH = 9.3 Day Dependent Spectra ... 188

Appendix I – Second Derivatives Plot, Individual Absorbance Spectra, and Tabulated Peak Positions of CO Incubated Native, E17Q, and R355K ... 189

Appendix J – References ... 191

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Inorganic Chemistry, Physical
Keyword	[NiFe]-hydrogenase Enzyme variants Transient absorption spectroscopy Mechanistic enzymology Metal-ligand cooperativity Infrared spectroscopy Photochemical reduction
Committee Chair / Thesis Advisor	R. Brian Dyer, Emory University
Committee Members	Tianquan Lian, Emory University Craig L. Hill, Emory University

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