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

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

Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(*)) in either the ferric or ferrous state and has efficient NO(*) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

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

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

Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the <i>Chlamydomonas reinhardtii</i> Hemoglobin THB1

Nitrate metabolism in <i>Chlamydomonas reinhardtii</i> involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3–4 × 10³ s^–1 at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by ¹⁵N_<i>z</i>-exchange spectroscopy are slow on the chemical shift time scale (10¹–10² s^–1 at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions

Histidine–Lysine Axial Ligand Switching in a Hemoglobin: A Role for Heme Propionates

The hemoglobin of <i>Synechococcus</i> sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When <i>b</i> heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent p<i>K</i>_a of 9.3 and a Lys-ligated population of ∼60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20–30 s^–1 by NMR exchange spectroscopy. Lys42–His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome <i>c</i>, in which Lys–Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only

Facile Heme Vinyl Posttranslational Modification in a Hemoglobin

Iron-protoporphyrin IX, or <i>b</i> heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the <i>c</i>-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium <i>Synechocystis</i> sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its <i>b</i> heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid <i>b</i>/<i>c</i> heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of <i>Synechocystis</i> GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117–2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine–heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection

Helix-Capping Histidines: Diversity of N–H···N Hydrogen Bond Strength Revealed by ^2h<i>J</i>_NN Scalar Couplings

In addition to its well-known roles as an electrophile and general acid, the side chain of histidine often serves as a hydrogen bond (H-bond) acceptor. These H-bonds provide a convenient pH-dependent switch for local structure and functional motifs. In hundreds of instances, a histidine caps the N-terminus of α- and 3₁₀-helices by forming a backbone NH···Nδ1 H-bond. To characterize the resilience and dynamics of the histidine cap, we measured the <i>trans</i> H-bond scalar coupling constant, ^2h<i>J</i>_NN, in several forms of Group 1 truncated hemoglobins and cytochrome <i>b</i>₅. The set of 19 measured ^2h<i>J</i>_NN values were between 4.0 and 5.4 Hz, generally smaller than in nucleic acids (∼6–10 Hz) and indicative of longer, weaker bonds in the studied proteins. A positive linear correlation between ^2h<i>J</i>_NN and the difference in imidazole ring ¹⁵N chemical shift (Δ¹⁵N = |δ¹⁵Nδ1 – δ¹⁵Nε2|) was found to be consistent with variable H-bond length and variable cap population related to the ionization of histidine in the capping and noncapping states. The relative ease of ^2h<i>J</i>_NN detection suggests that this parameter can become part of the standard arsenal for describing histidines in helix caps and other key structural and catalytic elements involving NH···N H-bonds. The combined nucleic acid and protein data extend the utility of ^2h<i>J</i>_NN as a sensitive marker of local structural, dynamic, and thermodynamic properties in biomolecules