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BRION ALLEN BERMAN Studies of a Novel Hemerythrin-Like Protein From BRION ALLEN BERMAN Studies of a Novel Hemerythrin-like Protein from the Anaerobic, Hyperthermophilic Archaeon, Methanococcus jannaschii (Under the Direction of DONALD M. KURTZ) Hemerythrin (Hr) is a non-heme iron protein, which originally had only been found in marine invertebrates where it functions as an oxygen-transport or storage protein. Hrs typically contain an oxo-/hydroxo-bridged non-heme diiron site surrounded by a four-helical bundle protein fold. All Hrs have seven conserved amino acids that furnish iron ligands, namely five histidines, one aspartate and one glutamate. Subsequently, homologues have been found in other organisms. Through protein and nucleotide searches, a previously undescribed protein having the Hr-like sequence motif (MjHr), was discovered in the Methanococcus jannaschii genome. MjHr exhibits structural and spectral properties resembling those of previously characterized Hrs. This Hr-like protein is the first to be described from archaea. Unlike Hrs that function as O2- carrying proteins, MjHr is proposed to function as an O2 sensor or scavenger within this methanogenic anaerobe. INDEX WORDS: Hemerythrin, Methanococcus jannaschii, O2-binding, Stopped- flow spectrophotometry, Electron paramagnetic resonance (EPR), Circular dichroism (CD), Western-blot, Metalloprotein, Reconstitution, Overexpression, Protein Purification STUDIES OF A NOVEL HEMERYTHRIN-LIKE PROTEIN FROM THE ANAEROBIC, HYPERTHERMOPHILIC ARCHAEON, METHANOCOCCUS JANNASCHII by BRION ALLEN BERMAN B.S., The University of South Alabama, 1999 A Thesis Submitted to the Graduate Faculty of The University Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2001 © 2001 Brion Allen Berman All Rights Reserved STUDIES OF A NOVEL HEMERYTHRIN-LIKE PROTEIN FROM THE ANAEROBIC, HYPERTHERMOPHILIC ARCHAEON, METHANOCOCCUS JANNASCHII by BRION ALLEN BERMAN Approved: August 7, 2001 Major Professor: Donald M. Kurtz Committee: Michael K. Johnson Robert S. Phillips Electronic Version Approved: Gordhan L. Patel Dean of the Graduate School The University of Georgia December 2001 IN MEMORY OF Milton “Pop” Kozlove (1917-1995) iv ACKNOWLEDGMENTS First, I would like to thank my family. I am indebted to my parents for their love, support, and pointing me in the right direction. I do not know what I would have done without the endless conversations and exhausted worrying from Mom. I undoubtedly would like to thank Dad for taking time to play golf and touring around the country with me, as well as, making sure I stay on top of things. I do not know if I could have stayed sane throughout graduate school without Jason taking me to different soccer tournaments and getting me outdoors. I especially would like to thank my grandparents, Jean Rose and Pop, for all the good times we spent together. I would like to thank Leigh for being supportive, understanding, and patient. I know it has not been easy. I do not know how I would have survived without the needed breaks from reality with the “Mexicali” crew: Cindy, Bryan, and Joe. It has been a fun time!!! I am grateful for Dr. Donald M. Kurtz, Jr. for his guidance and his financial support during my studies in his lab. I would also like to thank Drs. Robert Phillips and Michael Johnson for serving on my committee. I cannot begin to say enough about my labmates, Dr. Eric Coulter, Zanna Beharry, Joseph Emerson, Radu Silaghi, Shi Jim, and Dr. Kim Ng, for all the stimulating discussions, guidance, and moral support. Eric and Zanna, I do not know where to even start for all your help. I am deeply grateful. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.................................................................................................v CHAPTER 1. INTRODUCTION ....................................................................................................1 A. Oxygen-Carrying Proteins ............................................................................1 B. Properties of Dioxygen and Its Metal Complexes ........................................2 C. Properties of Hrs............................................................................................3 D. Hemerythrin-like Proteins From Other Organisms.......................................6 E. Goals of This Research..................................................................................7 2. EXPERIMENTAL..................................................................................................23 A. Cloning of the M. jannaschii Hr-like Protein .............................................23 B. Overexpression and Purification of Recombinant MjHr.............................25 C. Characterization of MjHr ............................................................................28 D. Preparation of Polyclonal Antibodies Against MjHr..................................33 E. Western Blot Analyses of Recombinant and Native MjHr .........................34 F. Kinetics of O2 Reactions with Recombinant deoxyMjHr............................35 3. RESULTS AND DISCUSSION.............................................................................39 A. Characterization of Recombinant MjHr......................................................39 B. Spectral Properties of Recombinant MjHr..................................................41 vi vii C. Exogenous Ligand Binding to MjHr...........................................................42 D. Activity of MjHr .........................................................................................44 E. Tests for Hydrogen Peroxide Production During Autoxidation of MjHr....45 F. Kinetics of O2 Reactions with deoxyMjHr..................................................45 G. Discussion ...................................................................................................47 REFERENCES ..................................................................................................................69 CHAPTER 1 INTRODUCTION A. Oxygen-Carrying Proteins There are three categories of O2-carrying proteins found in nature, all of which contain metal in their active sites. These oxygen-carrying proteins include the heme containing proteins, hemoglobin (Hb)/ myoglobin (Mb), the non-heme diiron proteins, hemerythrin (Hr)/ myohemerythrin (myoHr), and the copper-containing protein, hemocyanin (Hc). These proteins and homologues can be found in a wide variety of organisms ranging from vertebrates and invertebrates for Hb and Mb to arthropods and mollusks for Hc and to marine invertebrates and bacteria for Hr and myoHr. A rationale for the natural selection of iron and copper for reversible oxygen binding is the relativelyhigh bioavailabilities of both metals and their abilities to complex with dioxygen. The deoxy- and oxy- active site structures are shown in Figure 1-1 [1] and the protein backbone structures are shown in Figure 1-2 for all three types. Some properties of each of these proteins are compared in Table 1-1[2]. The most-studied and most naturally abundant of O2-carrying proteins are Hb and Mb. The active sites of hemoglobin and myoglobin (the monomeric analog of Hb) incorporate iron via a protoporphyrin IX (heme) group and a “proximal” histidine from the protein. The reduced forms of Hb and Mb have the heme in a five-coordinate high- spin Fe(II) complex. Dioxygen binds to the open, axial position on the iron in a bent, end-on fashion to the iron with 115-160° Fe-O-O bond angles (cf. Figure 1-1). The O2 is 1 2 formally reduced to the superoxide level, while the iron is formally oxidized to low-spin III - Fe(III). The Fe -O2 adduct is stabilized by hydrogen bonding to the “distal” histidine, which lies on the opposite side of the heme from the “proximal” histidine (not shown in Figure 1-1) [2]. The second most abundant O2-carrier is hemocyanin. Recently, X-ray crystal structures of the deoxy- and oxy- forms of L. polyphemus became available [3, 4]. In deoxyHc, there are two Cu(I) atoms each coordinated by three histidine residues. Upon oxygenation, the O2 is formally reduced to peroxide and binds in a side-on, bridging manner between the two Cu atoms, which are formally oxidized to Cu(II). The third class of O2-carrying proteins, hemerythrin and myohemerythrin (monomeric analogue of Hr) is found in a few marine invertebrates, such as sipunculids, brachiopods, priapulids, and annelids [5]. Despite the name, Hr does not contain a heme group, but rather a non-heme diiron site that reversibly binds oxygen. Since Hr is the focus of this research, its structure and function is discussed in greater depth below. B. Properties of Dioxygen and Its Metal Complexes In order to understand the ability of these proteins to bind dioxygen, some properties of dioxygen itself are first discussed. O2 is known to be reduced to superoxide - 2- (O2 ) or peroxide (O2 ) in biochemical systems. Dioxygen is readily reduced thermodynamically, but not kinetically. This difference between the thermodynamic and kinetic properties can be understood from the energy difference between the highest- occupied molecular orbitals (HOMO) and the lowest-unoccupied molecular orbitals (LUMO) of O2 (Figure 1-3) [1]. The slow kinetic reactivity can be rationalized because 3 O2 has a triplet ground state whereas organic molecules typically have singlet ground states, thus making them spin-forbidden from reacting with O2. Transition metal ions often have one or more unpaired d-electrons, and are therefore, not spin-forbidden from reacting with O2. Since transition metal-ions can have multiple positive
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