Graduation Semester and Year

Spring 2025

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Kayunta Johnson-Winters

Second Advisor

Kwangho Nam

Third Advisor

Subhrangsu Mandal

Fourth Advisor

Jongyun Heo

Abstract

F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH using reduced F₄₂₀ as the hydride donor. Both NADPH and the F₄₂₀ cofactor play significant roles in various metabolic pathways, including glycolysis and methanogenesis, particularly in methanogenic and sulfate-reducing archaea. These organisms play an essential role in global carbon cycling by converting carbon dioxide to methane through the methanogenic pathway, which is directly tied to energy production. This thesis investigates the hydride transfer mechanism of Fno from the extreme thermophile Archaeoglobus fulgidus, using fluorescence binding, steady-state and pre steady-state kinetics.

Chapter 1 outlines the structural and spectral characteristics of the F₄₂₀ cofactor and its phylogenetic distribution across diverse species. It also reviews the existing literature and prior studies on wild-type Fno (wtFno). Previous binding experiments revealed a Hill coefficient of 0.6 implying negative cooperativity binding. The steady-state kinetic experiments revealed a double reciprocal plot which was downward and concave in curvature, suggesting negative cooperativity kinetics. The pre steady-state kinetic analyses of wtFno revealed biphasic kinetics which is an initial faster burst phase followed by a slower phase. The amplitude of the burst phase indicated a 50% reduction of the F₄₂₀ cofactor, suggesting half-site reactivity. These data combined imply that Fno is involved in the regulation of intracellular NADPH levels. Key amino acids suspected of subunit communication due to their strategic positions at the interface include Arg186, Thr192, Ser190, and His133.

Chapter 2 describes the generation and characterization of Fno variants to probe the roles of Arg186, Thr192, Ser190, and His133 in inter-subunit communication. The Fno variants (R186Q, R186K, R186I, T192V, T192A, H133A, H133N, and S190A) were analyzed using fluorescence binding, steady-state and pre steady-state kinetic experiments, along with gel electrophoresis, and in-gel digestion. The results showed that several variants, R186Q, R186K, R186I, T192V, and S190A did not exhibit cooperativity, suggesting that Arg186, Thr192, and Ser190 are critical for subunit communication.

Building on previous work by the Johnson-Winters lab using the truncated F₄₂₀ precursor (FO), which showed negative cooperativity and half-site reactivity, Chapter 3 explores whether these effects are artifacts of the truncated cofactor. Experiments were conducted using both FO and F₄₂₀-2 across a pH range (5.8–7.8), reflecting the optimal growth range for A. fulgidus (most active at pH 6.5). The binding studies revealed that enzyme affinity for both cofactors remained in the nanomolar range but decreased with increasing pH. Notably, negative cooperativity was observed only between pH 6.3 and 6.5, disappearing outside this range. The steady-state kinetics revealed variable catalytic efficiency for F₄₂₀-2 across the pH spectrum, while the efficiency for FO remained relatively consistent. At pH 6.5, Fno was more efficient with FO, and kcat values for both cofactors were comparable within experimental error. Pre-steady-state data at pH 6.5 revealed biphasic kinetics with burst phase amplitudes of 40% and 49% for F₄₂₀-2 and FO, respectively. These findings confirm that half-site reactivity persists at this pH regardless of cofactor tail length, ruling out the truncated FO as a source of experimental artifact. In conclusion, this work elucidates the impact of cofactor structure and pH on Fno catalysis. The findings suggest that Fno employs cooperative mechanisms to modulate NADPH production and maintain the redox state of F₄₂₀ based on cellular needs.

Keywords

F420-dependent enzymes, Fno F420H2:NADP+ oxidoreductase

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

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