Investigating the metalloproteome of bacteria and archaea

Abstract

Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.