Looking Past the Active Site: Equilibrium and Kinetic Studies of a Perturbed Second Coordination Sphere Restricted; Files Only

Abstract

As greenhouse gas emissions continue to increase at an alarming rate, researchers are scrambling to find an alternative fuel source to decrease the mass of greenhouse gases that are being emitted each day with our large dependence on fossil fuels. In the search of alternative, renewable, carbon-neutral sources of fuel, hydrogen gas has earned its spotlight due to its carbon-free combustion reaction, the product of which is only water. However, the problem with hydrogen lies with its production. In 2021, 84% of the 94 million tons of hydrogen gas was produced by steam methane reforming, a process that produces 5.5 tons of CO2 for every ton of H2. To free hydrogen of its carbon ties, many avenues are being explored, one of which is to turn to nature for its evolution-driven solution: hydrogenase. Hydrogenase is an enzyme that catalyzes the reversible oxidation of H2, but many mechanistic and functional details of the second coordination sphere in hydrogenases are unknown. So, if the mechanistic and functional details of the protein scaffold can be fully characterized, it may be possible to develop novel and improved bio-inspired systems to produce carbon-neutral H2. To this end, the aim of this thesis is to investigate the effect of E17D and E17Q point mutations in soluble hydrogenase I from Pyrococcus furiosus (Pf SH1) to gain insight on the effect of perturbing the second coordination sphere of Pf SH1 via Fourier-transform and transient absorbance infrared spectroscopy. In conjunction with previous studies, our results indicate that the glutamic acid of the second coordination sphere in Pf SH1 greatly affects the catalytic landscape of the enzyme beyond the proton transfer chain.

Table of Contents

Chapter 1. Introduction and Motivation of Research...................................................................... 1

1.1. The climate crisis .................................................................................................................. 1

1.2. The hydrogen economy ........................................................................................................ 1

1.3. Nature’s solution: hydrogenases .......................................................................................... 3

1.4. Pyrococcus furiosus soluble hydrogenase I .......................................................................... 5

1.5. The proton transfer chain .................................................................................................... 8

1.6. The glutamic acid residue’s far-reaching effect ................................................................. 10

Chapter 2. Temperature-Dependent Auto-Oxidation ................................................................... 11

2.1. Motivation .......................................................................................................................... 11

2.2. Materials ............................................................................................................................ 12

2.3. Method ............................................................................................................................... 13

2.3.1. IR cell preparation for FT-IR spectroscopy ................................................................... 13

2.3.2. FT-IR instrumentation preparation .............................................................................. 14

2.3.3. FT-IR data collection ..................................................................................................... 14

2.4. Results and Discussion........................................................................................................ 15

2.4.1. FT-IR data processing ................................................................................................... 15

2.4.2. TDEP difference spectra ............................................................................................... 15

2.5. Conclusion .......................................................................................................................... 18

Chapter 3. CO Incubation and Photolysis ...................................................................................... 19

3.1. Motivation .......................................................................................................................... 19

3.2. Materials ............................................................................................................................ 20

3.3. Method ............................................................................................................................... 21

3.3.1. IR cell preparation for TA-IR ......................................................................................... 21

3.3.2. TA-IR instrumentation preparation .............................................................................. 21

3.3.3. Transient absorbance data collection .......................................................................... 22

3.4. Results and Discussion........................................................................................................ 23

3.4.1. TA-IR data processing ................................................................................................... 23

3.4.2. Transient absorbance traces ........................................................................................ 24

3.5. Conclusion .......................................................................................................................... 28

Appendix ....................................................................................................................................... 29

Appendix A. Exploded view of an IR cell .................................................................................... 29

Appendix B. IR cell assembly ..................................................................................................... 30

Appendix C. Fourier-transform infrared spectrometer desiccate chamber .............................. 30

Appendix D. Transient absorbance infrared instrumentation diagram ..................................... 31

References ..................................................................................................................................... 32

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Inorganic Chemistry, Physical
Keyword	Pyrococcus furiosus hydrogenase proton transfer chain second coordination sphere point mutation perturbation
Committee Chair / Thesis Advisor	Dyer, R. Brian, Emory University
Committee Members	Barba, David, Emory University Hill, Craig L., Emory University Liu, Fang, Emory University

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