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UBC Theses and Dissertations

Discovery and characterization of a new zinc-bacteriochlorophyll biosynthetic pathway and photosystem in a magnesium-chelatase mutant Jaschke, Paul Richard Ayre

Abstract

Porphyrins, for example heme and chlorophyll, are vital to biological processes such as respiration and photosynthesis. Both cofactors are synthesized through a common pathway to protoporphyrin IX (PPIX) which then branches: Fe²⁺ chelation into the macrocycle by ferrochelatase results in heme formation; by contrast, Mg²⁺ addition by Mg-chelatase commits the porphyrin to (bacterio)chlorophyll synthesis. The purple bacterium Rhodobacter sphaeroides is a model for bacteriochlorophyll a (BChl) biosynthesis and type-2 reaction center (RC) structure and function. While studying RC protein assembly it was discovered that a bchD (Mg-chelatase) mutant did not produce BChl as wild-type (wt) cells do, but instead produced small quantities of an alternative BChl in which Mg²⁺ was substituted by Zn²⁺. Zn-BChl α has been found in only one other organism before, the related acidophilic purple phototrophic bacterium Acidiphilium rubrum. The overall objectives of this thesis were two-fold: (1) to elucidate the Zn-BChl biosynthetic pathway; and (2) to utilize this biosynthesis as a tool to probe aspects of photosynthetic apparatus function. The biosynthetic pathway of Zn-BChl in the bchD mutant was found to begin at ferrochelatase, which efficiently chelated Zn²⁺ into PPIX. The resultant Zn-PPIX was utilized by the BChl-biosynthetic pathway, with metabolites early in the pathway accumulating, but with low Zn-BChl levels. Two novel intermediates I described, protoporphyrin IX monomethyl ester and divinyl-protochlorophyllide, contained Zn²⁺ instead of Mg²⁺. The demonstrated Zn-BChl biosynthetic pathway is a new way to make BChl and facilitates the further engineering of alternate forms of (Zn-)BChl. The RC in the bchD mutant (Zn-RC) was found to bind six Zn-BChls, instead of the four Mg-BChls and two bacteriopheophytins of the WT-RC. Spectroscopic examination showed that electron transfer (ET) in the Zn-RC occurs at approximately the same rate as in the WT-RC, despite substitution of Zn-BChl for bacteriopheophytin α. We showed preservation of ET was due to the unusual tetracoordination state of the Zn-BChls in the bacteriopheophytin site. This discovery allows refinement of ET rules within pigment-protein complexes by showing that the coordination state and conformation of cofactors can have an equally important role as the protein.

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