Redox Enzymes for Biocatalysis: From Asymmetric Hydrogenation to CO2 Reduction Público
White, David (Fall 2022)
Abstract
Biocatalysis is an integral aspect of society as enzymes are used to catalyze a broad range of industrial reactions with excellent efficiency and selectivity over traditional catalysts. Unfortunately, several drawbacks prevent enzymes from widespread application such as substrate scope and stability. Furthermore, the mechanisms of many enzymes are not fully understood preventing protein engineering or the development of better catalysts. This dissertation focuses on two aspects: the discovery of novel biocatalysts and deciphering the mechanistic details of potential biocatalysts.
Old Yellow Enzymes (OYE) are excellent biocatalysts for asymmetric hydrogenation, a key reaction for the synthesis of numerous industrially relevant products. Unfortunately, the substrate scope of these enzymes is limited with many possessing identical stereoselectivity. By exploring the natural diversity of the OYE family, new, novel enzymes were identified with enhanced catalytic activities and oppositive enantioselectivity. In addition, OYEs capable of desaturation, the reverse reaction, at ambient temperatures were also observed – further expanding the known reactions these enzymes can facilitate.
CO2 reduction and hydrogen production are important reactions for alternative energy sources and are catalyzed by the metalloenzymes carbon monoxide dehydrogenase (CODH) and hydrogenase. The full catalytic details of both enzymes are currently unknown preventing further application of these proteins as biocatalysts and the development of better, more efficient catalysts. For CODH, direct spectroscopic observation of CO in the active site has yet to be accomplished. To resolve this, the first photosystem incorporating CODH II from C. hydrogenoformans was developed with quantum yields 10-fold greater than previous CO2 to CO photoenzymatic systems. Infrared spectroscopy of the CO inhibited photosystem identified two IR frequencies corresponding to Ni-CO stretches in the active site. Time resolved spectroscopy indicated that both IR bands are involved in the catalytic cycle, indicating that two different bindings of CO to the Ni may be possible. For hydrogenase, the mechanism of reactivation from the oxygen-insensitive inactivated state was observed using photoreduction paired with IR spectroscopy. The [FeFe] hydrogenase from D. desulfuricans was observed converting from the Htran state to the resting Hox state, providing evidence for the previously proposed reactivation mechanism for the enzyme.
Combined this work seeks to overcome common obstacles limiting adoption of enzymes as biocatalysts, namely the lack of novel biocatalysts and understanding the mechanistic details underpinning difficult chemical transformations.
Table of Contents
Chapter 1: Introduction 1
1.1 - A Brief Overview of Biocatalysis 2
1.2 - Redox Enzymes and their Cofactors 3
1.3 - Asymmetric Hydrogenation via Old Yellow Enzymes 6
1.3.1 – The Old Yellow Enzymes 6
1.3.2 – The Other Old Yellow Enzymes 10
1.3.3 – Native Function and Beyond 10
1.4 - Carbon Monoxide Dehydrogenase at Catalysts for CO2 Reduction 12
1.4.1 – Artificial Photosynthesis 12
1.4.2 – The [NiFe] Carbon Monoxide Dehydrogenases 14
1.5 – Hydrogen Production using Hydrogenase 17
1.6 Aims and Scope of this Thesis 19
1.7 References 21
Chapter 2: Exploration of the Biocatalytic Potential of the Old Yellow Enzyme Family 43
2.1 – Introduction 44
2.2 – Results and Discussion 48
2.2.1 – Mapping the OYE Family using SSN 48
2.2.2 - Individual Cluster Analysis 51
2.2.3 - Phylogenetic Analysis and Reorganization of the OYE Family 53
2.2.4 - Selection of Novel OYE Sequences for In Vitro Screening 54
2.2.5 - Selection of Substrate Mixes, Reaction Conditions, and Characterized OYE Activity 55
2.2.6 - Overall OYE Activity and Cluster Specific Trends 57
2.2.7 - Interesting Biocatalytic Candidates of Mixes I-III 60
2.2.8 - Redox Potential and Other Explanations for Desaturase Activity 63
2.3 – Conclusions 65
2.4 - Materials and Methods: 68
2.4.1 - Generation of SSNs 68
2.4.2 - Generation of Phylogenetic Trees 68
2.4.3 - Determination of the Solubility of Selected OYEs 68
2.4.4 - Expression of OYE Sequences using Modified PURExpress 69
2.4.5 - Activity Assay Conditions for Novel OYE Sequences 69
2.4.6 - GC/MS Parameters 70
2.4.7 - Expression and Purification of Selected OYE Sequences 70
2.4.8 - Xanthine-Xanthine Oxidase Assay for OYE Redox Determination 71
2.5 – References 73
Chapter 3: Light-Triggered Investigations into the Mechanism of Carbon Monoxide Dehydrogenase 82
3.1 – Introduction 83
3.2 - Results and Discussion 87
3.2.1 – Development of an Efficient CODH II-based Photosystem for CO2 Reduction 87
3.2.2 - Infrared Absorption of CO Inhibited CODH 94
3.2.3 - Kinetics of Enzyme Reduction by Reduced Mediator 97
3.2.4 – Transient Absorption for Identified Ni-CO Stretches 99
3.3 – Conclusions 103
3.4 – Materials and Methods 105
3.4.1 - Synthesis and Preparation of CdSe/CdS Dot-in-Rods (DIR) 106
3.4.2 - Ligand Exchange of CdSe/CdS DIR 106
3.4.3 – Synthesis of the Mediator DQ53 107
3.4.4 – Enzyme Preparation and Purification 107
3.4.5 - GC Parameters and Calibration Curve for Detection of CO 108
3.4.6 - Standard Photoreduction Assay Conditions 109
3.4.7 - Quantum Efficiency Calculations 110
3.4.8 - Sample Preparation for Infrared Experiments 111
3.4.9 - Steady State CODH CO Inhibition FTIR Conditions 111
3.4.10 - Time Resolved Infrared Spectroscopy of CODH 112
3.5 – References 114
Chapter 4: Photoreduction of the Air Tolerant State in [FeFe] Hydrogenases 121
4.1 – Introduction 122
4.2 - Results and Discussion 124
4.2.1 - Light-Titrated Reduction of Inactivated DdHydAB 124
4.3 – Conclusions 127
4.4 - Materials and Methods 128
4.4.1 - Synthesis and Preparation of CdSe Nanorods 128
4.4.2 - Ligand Exchange of CdSe Nanorods 129
4.4.3 – Synthesis of PDQ 129
4.4.4 – Enzyme Preparation 129
4.4.5 - Sample Preparation for Infrared Experiments 129
4.4.6 - Steady State CODH CO Inhibition FTIR Conditions 130
4.5 – References 131
Chapter 5: Conclusions 134
5.1 – Conclusions 135
5.2 – Future Outlook 137
5.3 – References 138
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