Chemistry and Chemical Biology ETDs

Publication Date

4-16-2021

Abstract

Molybdenum (Mo) is an essential element that plays an important role in global nitrogen, carbon, and sulfur cycles with a critical role in human metabolism and ecological balance. It becomes catalytically active when complexed with the pyranopterin dithiolene ligand (PDT), forming the nearly ubiquitous molybdenum cofactor (Moco). The complex biosynthetic pathway of Moco, its presence in all three domains of life, and its role as a constituent cofactor in the last universal common ancestor (LUCA) all point toward the importance of the PDT in the development of life on Earth. Molybdoenzymes catalyze the two-electron oxidation or reduction of substrates that is usually coupled to an oxygen atom transfer. The PDT adopts several tautomeric and oxidation states in proteins, and this has been suggested to contribute to enzyme function through its ability to facilitate redox reactions by acting as an electron transfer conduit or a mediator of the enzyme redox potential.

In the following studies, the role of the PDT in electron transfer and catalysis is investigated. In the first of these studies, the potential role of the PDT as a directional electron transfer conduit during catalysis is assessed; in this, we have provided the first evidence that the PDT can function as biological unimolecular rectifier. Unimolecular rectifiers were first proposed by Aviram and Ratner over 40 years ago, but true rectification has been difficult to achieve, with RRs rarely exceeding 10. Our results indicate that the PDT can achieve RRs ~2-10, depending upon the tautomeric/oxidation state of the ligand and its relative connectivity to the electrodes. These results may elucidate how directional electron transfer is achieved in a biological system.

In the second of these studies, we sought an explanation for the conflicting crystallographic, kinetic, and spectroscopic data that has been published on the E. coli molybdoenzyme, MsrP. This enzyme functions as a periplasmic methionine sulfoxide reductase to “rescue” proteins damaged by oxidative stress from reactive chlorine species (RCS). The originally proposed active site of as-isolated MsrP, assigned by X-ray crystallography, is at odds with published spectroscopic data, and has led to the formulation of an unusual catalytic mechanism, in which the reducing equivalents necessary for catalysis are provided by the PDT ligand and not the metal. This mechanism, if correct, would represent a major paradigm shift from the accepted catalytic mechanisms of all other molybdoenzymes, in which the metal undergoes redox changes to provide the reducing equivalents necessary for catalysis. In this study, we provide compelling evidence that this mechanism and the originally proposed active site structure of as-isolated MsrP is incorrect. We provide evidence that as-isolated MsrP represents a thiol-inhibited species and reconciles the conflicting spectroscopic data. Lastly, we propose a new mechanism for the catalytic cycle of MsrP, in which the PDT does not undergo redox changes and the reducing equivalents are provided by Mo.

Language

English

Keywords

molybdenum, rectification, molybdoenzyme, MsrP, rectifier, YedY

Document Type

Dissertation

Degree Name

Chemistry and Chemical Biology

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Martin Kirk

Second Committee Member

Christopher Johnston

Third Committee Member

Brian Gold

Fourth Committee Member

Mark Walker

Download

Share

COinS