Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Matthew D. Liptak


Metalated tetrapyrroles, such as cobalamin (vitamin B12), catalyze a number of critically important biological processes including DNA synthesis, oxygen transport, methanogenesis, and photosynthesis. In addition, metalated tetrapyrroles are promising catalysts for non-biological energy transfer processes. Thus, their in vitro synthesis is an ongoing focus of research. These efforts have historically been limited by the metal insertion step, which is catalyzed by a class of enzymes called chelatases. Chelatases are highly selective toward their native tetrapyrrole and metal substrates, so artificial chelatases offer a more efficient and sustainable route to synthetic metalated tetrapyrroles. However, the origin of metal selectivity in chelatases remains poorly understood, which has hindered the development of artificial chelatases. Recent reports have targeted the affinity of the chelatase for available metals as a driver of metal selectivity, and that is explored herein.

Archaeoglobus fulgidus CbiXS and Methanosarcina acetivorans CfbA are archaeal chelatases responsible for catalyzing the insertion of cobalt and nickel, respectively, into sirohydrochlorin during the biosyntheses of cobalamin and cofactor F430. Furthermore, these chelatases are believed to be the evolutionary ancestors of larger chelatases such as the Salmonella cobalt chelatase CbiK and the human iron chelatase HemH. CbiXS and CfbA have high structural similarity, including several conserved active site residues, and are specific to the same tetrapyrrole substrate, but they natively bind and insert different metals. We have leveraged this by investigating their affinities for naturally abundant metals, and we have discovered starkly different metal affinities and binding ability between them in acidic environments. This was accomplished by spectroscopically measuring the dissociation constant for competitive metal chelators both with and without the proteins in solution. The dissociation constants between each metal and protein were extracted at pH 6, 7, and 8. The most significant differences were observed at pH 6. In an acidic environment, non-native metals are far more readily released. In neutral and basic environments, there is significantly less specificity for the native metal.

According to Kasha’s rule, electronic relaxation is only emissive from the first excited state. Zinc tetraphenylporphyrin (Zn TPP) and azulene are considered “anomalous” fluorophores because they radiatively relax from higher-lying excited states. Some anomalous fluorophores also experience increased emission intensity when aggregated, and one potential mechanism for this behavior is the suppression of Kasha’s rule (SOKR). Zn TPP, however, does not exhibit SOKR. The Photophysics of Zn TPP were explored computationally by manually constraining the conformation of the molecule and analyzing any differences in its electronic structure. There is no conformation corresponding to a conical intersection in Zn TPP like there is in SOKR fluorophores. In fact, the lowest excited states of a metalloporphyrin can never converge because of molecular symmetry. The spectroscopic qualities of azulene were thoroughly characterized in the 1950s, yet they remain difficult to computationally simulate. The ability of a wide array of computational methods to do this has been analyzed, and this has revealed the importance of electron correlation terms for modeling the electron structure of azulene and other non-alternant hydrocarbons. We have thus developed a method that more accurately models the absorptivity of the lowest two excited states of azulene than traditional methods.



Number of Pages

255 p.

Available for download on Thursday, April 03, 2025