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Investigations of Substrate Reduction by Nitrogenase Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2021 Investigations of Substrate Reduction by Nitrogenase: Light Powered Substrate Reduction by a CdS:FeMoco System and Understanding Dinitrogen Inhibition of Electron Transfer Hayden Kallas Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biochemistry Commons Recommended Citation Kallas, Hayden, "Investigations of Substrate Reduction by Nitrogenase: Light Powered Substrate Reduction by a CdS:FeMoco System and Understanding Dinitrogen Inhibition of Electron Transfer" (2021). All Graduate Theses and Dissertations. 8076. https://digitalcommons.usu.edu/etd/8076 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. INVESTIGATIONS OF SUBSTRATE REDUCTION BY NITROGENASE: LIGHT POWERED SUBSTRATE REDUCTION BY A CDS:FEMOCO SYSTEM AND UNDERSTANDING DINITROGEN INHIBITION OF ELECTRON TRANSFER by Hayden Kallas A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biochemistry Approved: Lance Seefeldt, Ph.D. Ryan Jackson, Ph.D. Major Professor Committee Member Alvan Hengge, Ph.D. D. Richard Cutler, Ph.D. Committee Member Interim Vice Provost for Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2021 ii Copyright © Hayden Kallas 2021 All Rights Reserved iii ABSTRACT Investigations of Substrate Reduction by Nitrogenase: Light Powered Substrate Reduction by a CdS:FeMoco System and Understanding Dinitrogen Inhibition of Electron Transfer by Hayden Kallas, Master of Science Utah State University, 2021 Major Professor: Lance Seefeldt, Ph.D. Department: Chemistry and Biochemistry The nitrogenase enzyme catalyzes the reduction of dinitrogen gas to ammonium and is responsible for making bioavailable approximately 50% of all biological ni- trogen. Nitrogenase is a two-component reductase consisting of a Fe protein that delivers electrons to the MFe (M = Mo, V, or Fe) protein where substrate reduction is catalyzed. Despite years of research into the subject, much is still unknown about the mechanism of substrate reduction and intramolecular electron transfer. Topics of interest include isolating and characterizing the independent reactivity of the active site cofactors and understanding the cause of dinitrogen inhibition of electron trans- fer rates. In this work, a novel method of studying the active site cofactor of the nitrogenase enzyme is proposed and its viability as a research tool is examined. CdS semiconductor nanoparticles were utilized to deliver electrons to the extracted active site cofactor of the Mo nitrogenase for substrate reduction. Kinetic and spectroscopic iv methods were used to characterize the CdS:cofactor catalytic system and test its fea- sibility for use as an investigative tool for nitrogenase cofactor research. Evidence of catalytically-active degradation products was uncovered by cofactor denaturation control experiments. This discovery largely eliminated the proposed system as a research tool. Nitrogen inhibition of electron transfer rates in the holoenzyme was studied us- ing multiple independent methods of enzyme kinetic measurements, evaluation of ammonium and hydrogen quantitation methods, and manipulation of nitrogenase re- action conditions affecting nitrogenase activity. Dinitrogen inhibition was found to only occur in kinetic measurements relying on product quantitation. Investigations of analytical methods resulted in the elimination of inaccurate product quantitation as a sole cause of dinitrogen inhibition. The extent of dinitrogen inhibition was found to be dependent on both pH and nitrogenase subunit ratio dependent but tem- perature invariant. The results presented here lead to the proposal of a novel and unaccounted-for product of nitrogenase catalysis under N2 as an explanation for these observations. (109 pages) v PUBLIC ABSTRACT Investigations of Substrate Reduction by Nitrogenase: Light Powered Substrate Reduction by a CdS:FeMoco System and Understanding Dinitrogen Inhibition of Electron Transfer Hayden Kallas Nitrogen fixation is a key step of the nitrogen cycle which makes biologically inert N2 gas available for organisms to use in the form of ammonia. Nitrogen fixing microorganisms all contain the same enzyme called nitrogenase which catalyzes the six electron transfers to N2 required for conversion into ammonia. Nitrogenase is a two-component enzyme that contains a cofactor composed of iron and sulfur as well as heavier metals whose identity can be molybdenum, vanadium, or an additional iron atom depending on the variant. The two components of nitrogenase are the MFe protein and the Fe protein. The Fe protein delivers electrons to the MFe protein where N2 conversion to ammonia takes place. Two outstanding topics of interest in the nitrogenase research field are (1) how the iron-sulfur active site cofactor of nitrogenase (FeMoco) interacts with the protein to facilitate electron transfer to sub- strates, (2) why electron transfer rates are sometimes slowed by N2 despite a long-held understanding that the rate of electron transfer is substrate invariant. The first question was addressed by using CdS nanoparticles to deliver electrons to the extracted active site cofactor of nitrogenase. This system allows the kinetics of substrate reduction at the cofactor to be investigated in order to understand what role the protein environment plays in reactivity. The CdS:cofactor system was char- acterized and its feasibility as an investigative tool was examined. The CdS:cofactor vi reductive system was found to be susceptible to decomposition resulting in the for- mation of multiple catalytic species; eliminating the current system as a mechanistic investigation tool. The second question was addressed by investigating what conditions and methods of measurement determine if N2 inhibition of electron transfer is observed. Several conditions were found to determine the extent of N2 inhibition observed including pH,nitrogenase subunit ratio, and the method by which electron transfer is measured. The results of these experiments led to the proposal of a novel, unaccounted-for product of nitrogenase catalysis under N2 as an explanation for N2 inhibition. vii ACKNOWLEDGMENTS The years I have spent in graduate school have taught me more about science and myself than any others of my life. I feel privileged to have been given the op- portunity to pursue scientific research at Utah State University and I thank all the following people for making this experience possible. My parents - There are no words that can accurately convey how grateful I am for your unwavering support and belief in my abilities. Thank you for supporting me in all of my endeavors. I truly could not have done this without you both. My sister - Hannah, you have always been there for me both as a sister and a friend. Having you to talk to has enriched both my graduate school experience and my life. My advisor - Dr. Seefeldt, thank you for both providing me the academic freedom to pursue topics of interest while also reining-in some of the crazier ideas. You’ve always struck an admirable balance between the many responsibilities of your position to the benefit of the lab. My labmates - You have been the largest influence in my graduate school ca- reer. I thank you all for your mentorship as well as providing an atmosphere of collaboration. My friends - Mathangi, Hannah, Ruidan, Kyle, Anna, and Spencer. The time spent with you guys has been a highlight of my graduate school career. Thank you all for not only being my comrades in the trenches but also for the inspiration and joy that you’ve brought into my life. Our collaborators - Thank you especially to Casey, Bryant, Kate, Jesse, and Luke for being the most pleasant and helpful research penpals (and instrument tech- nicians) that I could hope for. viii CONTENTS Page ABSTRACT ................................................ iii PUBLIC ABSTRACT .......................................... v ACKNOWLEDGMENTS ....................................... vii LIST OF FIGURES ........................................... x 1 INTRODUCTION .......................................... 1 1.1 Nitrogen Fixation ............................. 1 1.2 The Nitrogenase Enzyme ......................... 2 1.3 The Nitrogenase Mechanism and Kinetic Scheme ........... 3 2 Light Driven Substrate Reduction by a CdS:FeMoco Catalytic System ..... 11 2.1 Abstract .................................. 11 2.2 Introduction ................................ 11 2.3 Methods .................................. 15 2.3.1 Reagents and General Procedures ................ 15 2.3.2 Enzyme Growth and Purification ................ 15 2.3.3 Cadmium Sulfide Quantum Dot Synthesis ........... 16 2.3.4 Cadmium Sulfide Nanorod Synthesis .............. 16 2.3.5 FeMoco Extraction ........................ 17 2.3.6 Apo-MoFe Reconstitution .................... 18 2.3.7 EPR Spectroscopy ........................ 19 2.3.8 Nanoparticle-FeMoco Reactions ................. 19 2.4 Results and Discussion .......................... 20 2.4.1 Characterization of Extracted FeMoco ............. 20 2.4.2 Characterization of the CdS:FeMoco Catalytic System .... 22 2.5 Conclusions ................................ 29 2.6 Future Directions ............................
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