Structural Studies of Metalloproteins Using EPR Spectroscopy

Abstract

Metalloproteins play a central role in bacterial adaptation to anaerobic environments. The metal centers that these proteins contain enable diverse and complex chemical transformations, yet the mechanisms by which protein environments tune coordination geometry, redox properties, and substrate position to control metal cofactor reactivity remain incompletely understood. In two metalloenzymes, RquA and TrhP, these questions are particularly significant as each their chemical transformations dependent on metallocofactors that are atypical for these types of reactions. This thesis addresses this gap by defining the structure-function relationships of RquA and TrhP, with a focus on how the electronis structures of their metallocofactors may facilitate enzymatic activity. Chapter 1 provides a brief background of metalloprotein fundamentals, illustrating how spectroscopic and biochemical approaches can be used to probe properties like metal coordination, redox properties, and electronic structure. These concepts establish the framework for interpreting the results of the systems studied in the following chapters. Chapter 2 investigates RquA, a divalent metal cation dependent aminotransferase required for rhodoquinone biosynthesis. It had previously been shown that RquA was inactive in the absence of a divalent metal cation, and activity was restored when treated with Mn(II), Co(II), or Zn(II), yet the functional role and binding site of this divalent metal cation remained uncharacterized. Using EPR spectroscopy and other complementary techniques, we find that RquA binds these divalent metal cations in a 1:1 ratio. The coordination geometry of the metal is six-coordinate and is coordinated by one or more nitrogen-based ligands. These experimental results enabled us to propose a putative metal binding site of RquA. Chapter 3 studies TrhP, an iron-sulfur cluster dependent hydroxylase, responsible for the oxygen-independent hydroxylation of uridine 34 on certain bacterial tRNAs. While there have been \textit{in vivo} studies that have shown the necessity for four conserved cysteine residues for hydroxylation activity, there are no in depth \textit{in vitro} characterization studies of the identity of the iron-sulfur cluster that have been performed in strict anaerobic environments. Using UV-vis and EPR spectroscopy, we were able to show that TrhP's cluster nuclearity is preparation and redox dependent and is able to interconvert between the [2Fe-2S] and [4Fe-4S] state. Additionally, we were able to show that a low-potential ferredoxin YfhL is able to facilitate electron transfer to the clusters of TrhP. These findings provide a foundation for the understanding of the clusters TrhP binds in response to its redox environment.