HISTIDINE-HEME POSTTRANSLATIONAL MODIFICATION IN 2/2 HEMOGLOBINS: MECHANISM, CONSEQUENCES, AND ENGINEERING

Abstract

Proteins harness the intrinsic reactivity of heme to carry out a remarkable array of chemical tasks within the cell. Hemoglobins (Hbs) are heme proteins classically characterized by their reversible oxygen binding and storage function within the red blood cells of vertebrates. In the last twenty years, genomics initiatives lead to the discovery of genes encoding Hb in a wide variety of organisms including: plants, bacteria, cyanobacteria, fungi, and archaea. Phylogenetic studies indicate an ancient origin for the Hb superfamily and that Hbs preceded the cyanobacterial invention of photosynthesis and oxygenation of Earth’s atmosphere. Indeed, many microbial Hbs behave as enzymes and protect the cell by detoxifying reactive oxygen and reactive nitrogen species. As such, Hbs are a paradigm for protein functional diversification over evolutionary time. Hbs fold around their cofactor and via specific interactions tune heme reactivity to achieve functional chemistry. The genome of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. 6803 each encodes a single Hb (GlbN, 59% identity) with unusual structural features. In addition to the strictly conserved “proximal” histidine, these two GlbNs coordinate the heme iron using a 2nd histidine (“distal”). Bis-histidine hexacoordination is expected to lower reduction potential, accelerate electron transfer and condition ligand binding kinetics relative to classical “pentacoordinate” Hbs. GlbNs are further distinguished from other Hbs by their ability to bind heme covalently. The spontaneous irreversible modification links a non-coordinating histidine Ne2 to the heme 2-Ca and is analogous to the ubiquitous cysteine thioether (Cys-Sg-CaH-CbH3-heme) linkages observed in c cytochromes. The unusual His-heme posttranslational modification (PTM) in GlbN is thought to be triggered by reduction to the ferrous state. In vivo, GlbNs have been implicated in protecting their host organisms from reactive nitrogen species stress. Interestingly, both non-crosslinked and covalently modified (GlbN-A) proteins are detected in cyanobacterial cells. The following dissertation attempts to answer several questions regarding the unusual structural, chemical, and functional features of GlbNs. Chapter 1 introduces the reader to the diverse world of heme proteins and attempts to provide some background necessary to appreciate the GlbN results. Chapters 2-3 are NMR and UV-visible spectroscopic and protein engineering studies aimed at understanding the mechanism by which ferrous GlbN spontaneously converts to the covalently modified state (GlbN-A). In this work, we show that a cysteine can substitute for histidine in the GlbN covalent modification and also test several key mechanistic features of the electrophilic addition reaction. My most significant contribution includes demonstrating that reducing any amount of GlbN, in a pool of oxidized GlbN, will result in a facile electron transfer chain reaction ultimately converting the entire sample to GlbN-A. This observation constitutes a novel signal amplification mechanism for a heme protein. Additionally, we made (to my knowledge) the first quantitative measurement of electron self-exchange in a native form of Hb. Chapter 4 addresses differences between GlbN and GlbN-A with regard to their reactivity toward nitric oxide. In this work, I show that the covalent modification is required to prevent rapid heme dissociation upon binding nitric oxide in the ferrous state. Formation of the PTM also appears to extinguish the unusual nitric oxide reductase activity of GlbN. Chapters 5-6 are NMR structural studies in which we demonstrate that a histidine can be placed at different positions within the GlbN heme pocket and that nonnative PTM can be induced by reduction. We also show that the PTM can be successfuly transplanted in another Hb, in support that the covalent modification may be generally useful for heme protein engineering purposes. Chapter 7 uses sophisticated NMR methodologies to explore the structural consequences of native and non-native PTM within GlbNs. I demonstrate that hydrogen bond scalar couplings can be used to probe directly minute strain and relaxation in hydrogen bonding. I believe this work will be generally useful for understanding the biophysical chemistry of proteins, and also provides motivation for future studies of hydrogen bond perturbation within enzyme active sites. Overall, the results provide insight into the expanding chemical repertoire of the hemoglobin superfamily and pave a way for future structure-reactivity and protein engineering studies