Biochemical Studies of Regulatory Heme-Protein Interaction and Heme Regulation in Yeast

Abstract

Heme is a small biomolecule produced and used by living organisms from bacteria to humans. Heme consists of an organic porphyrin ring that coordinates an iron ion, Fe(II) or Fe(III), in its center. Heme can be in an oxidized or reduced state and is used extensively by organisms in a variety of redox reactions. As a cofactor in proteins of the electron transport chain, heme is crucial for cellular respiration and energy production. Heme is also the oxygen binding cofactor in globins such as hemoglobin and myoglobin, allowing organisms to distribute oxygen. Free heme in the cell is capable of producing reactive oxygen species, which are toxic to the cell. However, the degradation product of heme, bilirubin, is a powerful protectant against oxidation. In cytochromes, such as P450s and cytochrome c, heme is used in the synthesis and degradation pathways of sterols, lipids, and neurotransmitters. With such a central role in energy production, synthesis and degradation, oxidative stress, and oxygen transport, it makes sense that heme also acts as a very important signaling molecule regulating many genetic pathways such as oxidative stress response and carbon metabolism. The function of heme as a prosthetic group in proteins, such as cytochromes, is now well documented. Less is known, however, about its role as a regulator of metabolic and energetic pathways. This is due in part to some inherent difficulties in studying heme. Due to its slightly amphiphilic nature, heme is a “sticky” molecule that can easily bind non-specifically to proteins. In addition, heme tends to dimerize, oxidize, and aggregate in purely aqueous solutions; therefore, there are constraints on buffer composition and concentrations. Despite these difficulties, our knowledge of heme’s regulatory role continues to grow. This work describes the application of common molecular biology techniques to the unique situation of studying heme-protein regulation. Chapter 2 describes our findings of the heme regulation of Gis1, a yeast transcription factor and demethylase. Gis1 regulates the response to metabolic stress after the diauxic shift. Our lab previously identified Gis1 as a fast responder to hypoxia and re-oxygenation. Heme is closely related to oxygen utilization and signaling, and the Gis1 sequence contains two heme regulatory motifs (HRM), therefore we decided to study the regulation of Gis1 by heme. It was found that heme bound to at least two locations on Gis1, and that the zinc finger domain, which contains an HRM, promoted heme activation of Gis1 transcriptional and demethylase activities. The Jumonji domain, which contains the second heme binding site and an HRM, did not convey heme regulation of Gis1, but it did cause heme regulation of a different transcriptional activating domain. As a member of the JMJD2b/KDM4 family of demethylases, conserved in mammals, Gis1 represents a new class of heme signaling protein.