Proton, Electron and Proton-coupled Electron Transfer Dynamics in the Catalytic Mechanism of [NiFe] and [FeFe] Hydrogenases Pubblico

Abstract

Hydrogenases are enzymes which catalyze the reversible reduction of protons to molecular hydrogen with high kinetic rates and low over-potentials using earth abundant metals, iron and nickel. Their chemical mechanism is of significant interest as a model for biomimetic systems aimed at improving existing proton reduction catalysts, but their chemical mechanism has been elusive. In this thesis, kinetic methods, both based on traditional steady state techniques as well as novel pre-steady state methods, are explored and utilized to directly observe chemical changes during catalysis by a [NiFe] hydrogenase from the hyperthermophilic Pyrococcus furiosus as well as an [FeFe] hydrogenase from the hyperthermophilic Thermotoga maritima. Steady state kinetic analysis of the Pyrococcus furiosus [NiFe] hydrogenase was used to probe the rate determing steps and thermodynamics of redox mediator interactions revealing a product release rate determining step and relatively slow intramolecular proton transfer. Despite significant insight gained through steady state kinetic analysis, little information could be determined beyond the rate determining steps. To address this, methods for rapidly initiating the proton reduction activity of model hydrogenases were explored using photo-initiated electron transfer from small molecule photo-ionization, redox dye and quantum dot photo-sensitization. Once methods for photo-initiation were established, pre-steady state catalytic initiation of the Pyrococcus furiosus hydrogenase was investigated by time resolved spectroscopy, sensitive to the enzyme active site, revealing rich chemical dynamics on timescales 10⁴ s^-1 faster than the rate determining step(s). The active site dynamics probed by time resolved infrared transient absorbance demonstrated the kinetic validity of several reaction intermediates previously proposed based on equilibrium structural and spectroscopic data. Furthermore, the mechanism of the chemical reactions necessary for inter-conversion of the verified intermediates was elucidated, implicating various proton-coupled reduction pathways involved in enzymatic turnover. Additionally, pre-steady state investigation of the proton reduction activity of the Thermotoga maritima [FeFe] hydrogenase also revealed multiple chemical steps occurring faster than the overall catalytic rate and verified previously proposed catalytic intermediates as being kinetically competent.

Table of Contents

Chapter 1 - Introduction

1.1 - Renewable energy challenges, chemical fuels and the role of bioinorganic/biomimetic chemistry

1.2 - H2ases: Cellular function and implications on mechanism

1.2.1 - [NiFe] H2ases

1.2.2 - [FeFe] H2ases

1.3 - H2ases chemical mechanism

1.3.1 - [NiFe] H2ases

1.3.2 - [FeFe] H2ase

1.4 - Practical applications of fast kinetic analysis

1.5 - Hypothesis and scope of this thesis

1.6 - References

Chapter 2 - Experimental Methodology

2.1 - Introduction

2.2 - Enzyme expression and purification

2.2.1 - Thiocapsa roseopersicina [NiFe] H2ase

2.2.2 - Pyrococcus furiosus SHI

2.2.3 - Thermotoga maritima [FeFe] H2ase

2.3 - Materials

2.3.1 - Buffers

2.3.2 - Nanocrystal Synthesis

2.3.2.1 - CdTe Quantum Dots

2.3.2.2 - CdSe@CdS Quantum Dot in Rods

2.3.3 - Miscellaneous

2.4 - Analytical Methods

2.4.1 - UV-Vis

2.4.2 - FTIR

2.4.3 - Gas Chromatography

2.4.4 - Photoluminescence

2.4.5 - Time Resolved Photoluminescence

2.4.6 - Tandem Transient Infrared/Visible Absorbance

2.4.7 - Raman Detection of HD

2.5 - Enzyme Assays

2.5.1 - H2 Production

2.5.2 - H/D Exchange

2.5.3 - S⁰ Reduction

2.5.4 - CO Inhibition

2.5.5 - Temperature dependent MV⁺/MV²⁺ equilibrium

2.6 - Data analysis

2.6.1 - Transient Kinetics Fitting

2.7 - References

Chapter 3 - Steady state kinetic investigations of H2ase mechanism.

3.1 - Introduction

3.2 - Results and Discussion

3.2.1 - Proton Reduction: The Role of Proton Transport

3.2.2 - Active Site Chemistry of Proton Reduction

3.2.3 - Equilibrium and Enzyme Bias

3.3 - Conclusions

3.4 - References

Chapter 4 - Rapid Initiation of H2ase Activity Through Photo-chemistry.

4.1 - Introduction

4.2 - Results and Discussion

4.2.1 - Tris(bipyridine)ruthenium(II) photo-sensitization

4.2.2 - CdTe Quantum Dot Photo-Sensitization

4.2.3 - On Nanoparticle-Enzyme Orientation Effects.

4.2.4 - NADH Photoionization

4.3 - Conclusions

4.4 - References

Chapter 5 - Pre-Steady State Dynamics of H2ases

5.1 - Introduction

5.2 - Results and Discussion

5.2.1 - Steady State Spectroscopic Characterization of Pf

[NiFe] SHI

5.2.2 - Photogeneration of e^-aq and MV⁺ Observed by Time

Resolved Visible Spectroscopy

5.2.3 - Photo-Reduction of MetMb: A Case Study

5.2.4 - Photo-Reduction of Pf SHI

5.2.5 - Mechanistic Aspects of Proton Reduction by Pf SHI.

5.2.6 - Proton Reduction by the [FeFe] H2ase from

Thermotoga maritima.

5.3 - Conclusions

5.4 - References

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Physical
Parola chiave	Kinetics Time Resolved Infrared Proton-coupled Electron Transfer Hydrogenase Electron-proton Transfer Spectroscopy
Committee Chair / Thesis Advisor	Dyer, Brian, Emory University
Committee Members	Lian, Tim, Emory University MacBeth, Cora E., Emory University

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