Date of Award
Summer 2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Antony, Edwin
Second Advisor
St. Maurice, Martin
Third Advisor
Fiedler, Adam
Abstract
Iron-Sulfur ([Fe-S]) clusters are the most common transient electron carriers in cells and are necessary for basic metabolism of all life. Bacterial systems use two operons (isc and suf ) for the biogenesis and delivery of [Fe-S] clusters to various proteins. Once delivered, they serve as transient electron carriers necessary for both heterotrophic and autotrophic metabolism and reduction/oxidation chemistry. This work utilizes the hetero-octameric Dark operative Protochlorophyllide Oxido-Reductase (DPOR) complex as a platform to investigate [Fe-S] cluster biogenesis and the concerted action of its multiple [Fe-S] clusters and protein subunits. DPOR catalyzes the penultimate step in bacterial chlorophyll synthesis and accounts for more than half of the chlorophyll produced on earth. DPOR stereo-specifically reduces the C17-C18 double bond of protochlorophyllide (Pchlide) to yield chlorophyllide (Chlide). This reduction requires 2 cycles of electron transfer and the hydrolysis of 4 ATP molecules. The DPOR complex is composed of two separable components, an ATPase electron donor containing one [4Fe-4S] cluster (BchL) and a catalytic electron acceptor containing 2 [4Fe-4S] clusters (BchNB.) BchL exists as a stable alpha-2 homodimer, and BchNB exists as a stable alpha-2 beta-2 heterotetramer, containing two [4Fe-4S] clusters that accept electrons from BchL and are terminally donated to one of two Pchlide molecules bound to BchNB. The overall complex is arranged as two identical catalytic halves. In the presence of ATP, BchNB forms a transient complex with BchL and electron transfer to Pchlide is coupled to ATP hydrolysis. The architecture of DPOR is evolutionarily conserved, with the most notable examples being the next protein in the chlorophyll synthesis pathway Chlorophyllide Oxidoreductase (COR), and nitrogenase which share the same stoichiometry of subunits. The quaternary structure of DPOR has two pseudo C2 axes of symmetry, one bisecting the BchL dimer, and one bisecting the BchNB tetramer. This complexity makes study of these enzymes complex but the degree of homology and conservation in these life critical enzymes begs the questions why does DPOR function in such a complicated manner? Is this complexity even necessary, and how is information communicated across the long distances between subunits of DPOR? The specific role of individual events from binding of substrate (whether ATP or Pchlide), electron transfer events, and identical halves of BchL or BchNB function remains unresolved for DPOR. This document details a classical biochemical approach that reveals mechanistic principles underlying the structural and functional complexity of DPOR. The main findings of this work are 1) proof of the non-redundancy of the isc and suf operons in the heterologous expression of proteins, 2) novel findings on the role of ATP in the BchL homodimer unique to DPOR, and 3) the sequential electron transfer in the BchNB heterotetramer. Overall it indicates that identical halves of DPOR components accomplish complex long-range allosteric communication between symmetrical structures that requires two functional identical halves for overall activity, that key features of DPOR are evolutionarily maintained because they regulate key steps that must work in harmony to accomplish the symphony of steps that allow DPOR and related enzymes to function.