Date of Award:

5-2016

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Chemistry and Biochemistry

Department name when degree awarded

Biochemistry

Committee Chair(s)

Edwin Antony

Committee

Edwin Antony

Committee

Korry Hintze

Committee

Lance Seefeldt

Abstract

Chlorophylls are essential pigment molecules that function in photosynthesis, and serve to aid in utilizing energy from sunlight to power cellular processes in plants, and other organisms. To make chlorophyll, photosynthetic organisms devote an abundance of resources and energy to ensure their appropriate construction. This process of making chlorophylls is highlighted by the penultimate step in the pathway—the conversion of protochlorophyllide (Pchlide) to chlorophyllide a (Chlide).

This conversion can be mediated in two different ways, depending on the type of organism. The first method incorporates the use of a light-activated system called the light-dependent protochlorophyllide oxidoreductase (LPOR). This system, as the name suggests, uses light to trigger the production of chlorophylls for use in photosynthesis. The focus of research provided hereafter is centered on a structurally unrelated dark-operative system (DPOR), which generates chlorophyll in the absence of light, or in lowlight conditions.

DPOR is structurally related to the enzyme nitrogenase, which functions to reduce atmospheric nitrogen into a form usable for living systems to incorporate into their metabolism. Both DPOR and nitrogenase are similar in that both require ATP binding and hydrolysis for electron transfer to occur, but differ in that DPOR is a much slower enzyme. Even though ATPase activity is essential for catalysis, the role of ATP throughout the catalytic mechanism is not well understood. The research contained herein was conducted in order to better characterize the role of ATP in DPOR during this critical step in chlorophyll production.

When comparing steady-state rates between DPOR and nitrogenase, DPOR was found to perform 400-fold slower with respect to ATPase activity. However, the initial rates of ATP hydrolysis were found to be very similar, indicating that the two systems are divergent after the initial hydrolysis occurs. Mutational studies further show that both ATPase sites are required for normal function.

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