Rate constants for ligand binding to several wild type hemoglobins [21, 37].
Abstract
Cyanobacteria are oxygenic photosynthetic prokaryotes, practically present in every plausible environment on the earth. In 1996, the first cyanobacterial genome was sequenced from Synechocystis sp. PCC 6803 and the cyanobacterial genome database has been continuously growing with genomes from more than 300 cyanobacterial and other related species, so far. Synechocystis sp. PCC 6803 is one of the best-characterized cyanobacteria and has developed into a model cyanobacterium that scientists are using throughout the world. At the same time, the field of hemoglobin was undergoing a breakthrough with the identification of new globins in all three kingdoms of life including cyanobacteria. Since then, the newly identified globins in the cyanobacteria are raising intriguing questions about their structure and physiological functions, which are quite different from vertebrate’s hemoglobin and myoglobin. These hemoglobins have displayed unprecedented stability, unique heme coordination, novel conformational changes, and other properties that are not often observed in the globin superfamily. This chapter provides an overview of the unique globin from Synechocystis sp. PCC 6803, its interacting protein partners, proposed functions, and its biotechnological implications including potential in the field of artificial oxygen carriers.
Keywords
- Cyanobacteria
- Synechocystis sp. PCC 6803 hemoglobin
- Structural features
- Heme stability
- Physiological function
- Biotechnological application
1. Introduction
The ancient cyanobacteria played a fundamental role in changing the composition of the early, oxygen-poor reducing atmosphere into an oxidizing atmosphere of the earth. These tiny oxygenic phototrophs inhabit varied ecosystems and habitats ranging from oceans to hot springs and deserts [1]. They can also be found in extreme environments, such as acidic bogs and volcanoes. The plethora of available information on the diversity and physiology of cyanobacteria provides an excellent base for exploring their application in biotechnology. Because of their ability to harvest solar energy and convert atmospheric CO2 to useful products like biofuels and bioactive compounds, they serve as a promising organism which is used for medical treatments and various industrial applications [2].
Oxygen provides an enormous source of energy for biological functions; however, it can also be toxic to organisms. It is believed that cyanobacteria were among the earliest prokaryotic organisms responsible for the oxygen-rich environment on the young planet earth and currently, nearly 99% of the oxygen is contributed by the eukaryotic algae [3]. It has been revealed that all the eukaryotic phototrophic organisms derived the ability to produce oxygen during photosynthesis through endosymbiosis [4]. Later, it was discovered that the heme-containing protein that sequesters and protects the primitive cyanobacteria cells from toxic O2 is almost identical to the energy-generating apparatus in the photosynthetic bacteria [5]. The basic chemical apparatus became increasingly complex through time and evolution, however, the interaction between the metal atom in the porphyrin ring and the oxygen remains unchanged [6]. The heme-containing proteins form a large class of macromolecules that have diverse and distinct biological functions. Comparative analysis of hemoproteins revealed that the changes in the amino acid sequence and their interaction with the porphyrin ring mainly involved in the multitude of different functions which include electron-transferring cytochromes, intracellular peroxidases, and lignin-degrading extracellular peroxidases. The well-studied heme-binding proteins are vertebrate hemoglobin (Hb) and myoglobin (Mb). The heterotetrameric hemoglobin is present at high concentration (15 g/100 ml) in normal human blood and involved in the oxygen transport in the circulatory system whereas myoglobin is a monomeric oxygen storage protein mainly located in the cardiac and striated muscles [7].
Recent breakthroughs in molecular biology tools and genome sequencing techniques led to the identification of hemoglobin genes in almost all kingdoms of life including plants, animals, bacteria, and fungi. Extensive bioinformatics surveys of available genomes identified putative globins with several characteristics that are distinct from the classical globins [8]. These distinct globins have been designated as “novel” or “new” globins to distinguish them from the traditional globins. These new Hbs display differences in the coordination chemistry of heme Fe atom in which all six coordination sites are occupied in the absence of exogenous ligand and referred to as “hexacoordinated hemoglobins (HxHbs)” compared to “pentacoordinated” heme Fe coordination chemistry of classical vertebrate Hb and Mb [9, 10]. Another set of novel hemoglobins have been discovered which are 20–40 amino acid residues shorter than the mammalian hemoglobins, resulted in modification of the canonical “3-on-3” globin fold and provided them with a shortened “2-on-2” globin fold [11, 12, 13]. These classes of hemoglobins are called “truncated hemoglobins (TrHbs)” and constitute a major class of the globin family [14].
In 1992, the identification of truncated hemoglobin in the nitrogen (N2)-fixing cyanobacterium
2. Synechocystis sp. PCC 6803 hemoglobin: an unusual hemoglobin from a cyanobacterium
S6803 was the third unicellular prokaryotic and first non-diazotrophic photosynthetic organism whose genome is completely sequenced. The genome sequence analysis showed the presence of a single hemoglobin gene (coded by slr2097 gene; named as glbN), encoding 123- amino acid polypeptide chain sharing 55% sequence identity with the cyanoglobin from
2.1 Biochemical and structural features of Synechocystis Hemoglobin
In the year 2000, two different groups reported the preliminary biochemical characterization by cloning and over-expressing the slr2097 globin gene in
2.2 Role of key residues in the stability and folding of Synechocystis Hemoglobin
Preliminary investigation of
2.3 Ligand binding kinetics in Synechocystis Hb
The reaction of
Hb | k’O2 (μM−1 s−1) | kO2 (s−1) | k’CO2 (μM−1 s−1) | kentry, CO (μM−1 s−1) |
---|---|---|---|---|
240 | 0.014 | 90 | ||
390 | 79 | 41 | ||
25 | 0.2 | 6.8 | ||
Rice Hb (rHb1) | 68 | 0.038 | 6 | |
Sperm whale Mb | 17 | 15 | 0.5 | 17 |
Human Hb α- chain | 23 | 11 | 2.9 | 11 |
Human Hb β- chain | 79 | 28 | 7.1 | 11 |
Soybean LegHb (Lba) | 130 | 5.6 | 17 | 320 |
3. Proposed physiological function of Synechocystis hemoglobin
The new class of Hb differs from the classical Hbs in their cellular location, primary sequence, expression pattern, three-dimensional fold, heme pocket architecture, ligand binding characteristics, heme pocket electrostatics etc. and thus, forced the researchers worldwide to re-investigate their functions which might diverge widely during evolution of globins [11, 44]. Based on several reports, the oxygen transport and storage function which are usually associated with hemoglobins have been ruled out and various other functions including detection, scavenging, and detoxification of O2 and O2- derived species (e.g. NO and CO) have been proposed.
Despite numerous efforts, physiological function for
4. Biotechnological applications of Synechocystis hemoglobin
4.1 In designing a stable hemoglobin-based blood substitute using protein engineering approach
In the last few decades, there is a significant progress in the development of oxygen-carrying blood substitutes. Since the 1980s, human blood substitutes have been in the pipeline in the medical and life science research fields [52]. Currently, there are none in the market because of scientific and political reasons. There are a few blood substitutes still progressing through clinical trials, and the academic community is still actively improving the products, also known as oxygen therapeutics and hemoglobin-based oxygen carriers [53, 54]. Over the last few years, studies have focused on developing “recombinant hemoglobin-based oxygen carriers” (rHBOCs) which can be used as an alternative to blood during transfusion therapy. Recombinant human hemoglobin is produced in heterologous expression systems like
The newly discovered truncated and hexacoordinate globins exhibit unique features that allow the exploration of a whole range of proteins, some of which might be more stable than Mb, thus allowing newer ways for comparative mutagenesis strategies to improve stability.
4.2 Synechocystis hemoglobin as a fusion tag for enhancing the expression, solubility and purification of other proteins
Recent years have witnessed tremendous increase in the number of tags and the development of fusion strategies to facilitate the expression, purification, and solubilization of recombinant proteins which can be used as industrial enzymes, for drug discovery, and biotherapeutics [63]. There are now a wide variety of fusion tags available in the market which are well-characterized and used in the biotechnological industry to obtain highly purified biologically active recombinant proteins [64]. However, these available tags have a major limitation, i.e., the absence of any color to facilitate the visualization of target protein during the expression and purification process. Hemoglobins because of their distinctive bright red color, high solubility and stability offer a unique advantage of tracking of the target fused protein during expression and at different steps of purification and even in crystallization and thus minimizing the cost and time in fusion protein technology. Previous reports showed the use of visible tag systems such as flavoenzymes and hemeproteins that contain colored chromophores [65]. The
5. Conclusion
In the last few decades, an intense research effort from several researchers worldwide has enabled us to uncover the unique properties of
Acknowledgments
Suman Kundu acknowledge the financial assistance from Life Science Research Board, Defense Research and Development Organization (LSRB-DRDO; Grant ID O/o DG (TM)/81/48222/LSRB317/SH&DD/2017) New Delhi, India, University of Delhi (R&D grant), Department of Science and Technology (PURSE), Government of India and University Grants Commission (SAP grant), and Department of Biotechnology, Government of India. Mohd. Asim Khan acknowledges LSRB-DRDO, for Senior Research Fellowship (SRF). Sheetal Uppal is thankful to National Eye Institute, NIH for providing financial assistance.
Abbreviations
GlbN | Cyanoglobin |
HxHbs | Hexacoordinate hemoglobin |
FHb | Flavohemoglobin |
Hb | Hemoglobin |
SynHb | Synechocystis hemoglobin |
NMR | Nuclear magnetic resonance |
NOD | Nitric oxide dioxygenase |
nsHb | Non-symbiotic hemoglobin |
PDB | Protein data bank |
TrHb | Truncated hemoglobin |
O2 | Oxygen |
CO | Carbon monoxide |
NO | Nitric oxide |
HBOCs | Hemoglobin based oxygen carriers |
References
- 1.
Pisciotta JM, Zou Y, Baskakov IV. Light-dependent electrogenic activity of cyanobacteria. PloS one. 2010;5(5):e10821. - 2.
Singh S, Kate BN, Banerjee UC. Bioactive compounds from cyanobacteria and microalgae: an overview. Critical reviews in biotechnology. 2005;25(3):73-95. - 3.
Kasting JF, Siefert JL. Life and the evolution of Earth's atmosphere. Science. 2002;296(5570):1066-1068. - 4.
Margulis L. Symbiosis in cell evolution : microbial communities in the Archean and Proterozoic eons. San Francisco, 2 ed: New York (N.Y.) : Freeman; 1993. - 5.
Goldhor S. The History of Cell Respiration and Cytochrome. The Yale Journal of Biology and Medicine. 1966;39(3):207. - 6.
Hardison R. The evolution of hemoglobin: studies of a very ancient protein suggest that changes in gene regulation are an important part of the evolutionary story. American Scientist. 1999;87(2):126-137. - 7.
Hardison R. Hemoglobins from bacteria to man: evolution of different patterns of gene expression. Journal of Experimental Biology. 1998;201(8):1099-1117. - 8.
Vinogradov SN, Tinajero-Trejo M, Poole RK, Hoogewijs D. Bacterial and archaeal globins - a revised perspective. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2013;1834(9):1789-800. - 9.
Burmester T, Ebner B, Weich B, Hankeln T. Cytoglobin: a novel globin type ubiquitously expressed invertebrate tissues. Molecular biology and evolution. 2002;19(4):416-421. - 10.
Trent III JT, Watts RA, Hargrove MS. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. Journal of Biological Chemistry. 2001;276(32):30106-30110. - 11.
Milani M, Pesce A, Ouellet Y, Ascenzi P, Guertin M, Bolognesi M. Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme. The EMBO journal. 2001;20(15):3902-3909. - 12.
Moens L, Vanfleteren J, Van de Peer Y, Peeters K, Kapp O, Czeluzniak J, et al. Globins in nonvertebrate species: dispersal by horizontal gene transfer and evolution of the structure-function relationships. Molecular biology and evolution. 1996;13(2):324-333. - 13.
Pesce A, Couture M, Dewilde S, Guertin M, Yamauchi K, Ascenzi P, et al. A novel two-over-two α-helical sandwich fold is characteristic of the truncated hemoglobin family. The EMBO journal. 2000;19(11):2424-2434. - 14.
Kumar A, Nag M, Basak S. Truncated or 2/2 hemoglobins: a new class of globins with novel structure and function. Journal of proteins and proteomics. 2013;4(1):45-64. - 15.
Potts M, Angeloni SV, Ebel RE, Bassam D. Myoglobin in a cyanobacterium. Science. 1992;256(5064):1690-1692. - 16.
Kaneko T, Tabata S. Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803. Plant and Cell Physiology. 1997;38(11):1171-1176. - 17.
Scott NL, Lecomte JT. Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803. Protein Science. 2000;9(3):587-597. - 18.
Couture M, Das TK, Savard PY, Ouellet Y, Wittenberg JB, Wittenberg BA, et al. Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket. European journal of biochemistry. 2000;267(15):4770-4780. - 19.
Hoy JA, Kundu S, Trent JT, Ramaswamy S, Hargrove MS. The crystal structure of Synechocystis hemoglobin with a covalent heme linkage. Journal of Biological Chemistry. 2004;279(16):16535-16542. - 20.
Trent III JT, Kundu S, Hoy JA, Hargrove MS. Crystallographic analysis of Synechocystis cyanoglobin reveals the structural changes accompanying ligand binding in a hexacoordinate hemoglobin. Journal of molecular biology. 2004;341(4):1097-1108. - 21.
Nothnagel HJ, Love N, Lecomte JTJ. The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin. Journal of inorganic biochemistry. 2009;103(1):107-116. - 22.
Uppal S, Salhotra S, Mukhi N, Zaidi FK, Seal M, Dey SG, et al. Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin. Journal of Biological Chemistry. 2015;290(4):1979-1993. - 23.
Sturms R, DiSpirito AA, Fulton DB, Hargrove MS. Hydroxylamine reduction to ammonium by plant and cyanobacterial hemoglobins. Biochemistry. 2011;50(50):10829-10835. - 24.
Sturms R, DiSpirito AA, Hargrove MS. Plant and cyanobacterial hemoglobins reduce nitrite to nitric oxide under anoxic conditions. Biochemistry. 2011;50(19):3873-3878. - 25.
Kundu S, Premer SA, Hoy JA, Trent III JT, Hargrove MS. Direct measurement of equilibrium constants for high-affinity hemoglobins. Biophysical Journal. 2003;84(6):3931-3940. - 26.
Smagghe BJ, Trent JT, 3rd, Hargrove MS. NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo. PLoS One. 2008;3(4):e2039. - 27.
Preimesberger MR, Wenke BB, Gilevicius L, Pond MP, Lecomte JTJ. Facile heme vinyl posttranslational modification in a hemoglobin. Biochemistry. 2013;52(20):3478-3488. - 28.
Falzone CJ, Christie Vu B, Scott NL, Lecomte JT. The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state. J Mol Biol. 2002;324(5):1015-1029. - 29.
Vu BC, Jones AD, Lecomte JT. Novel histidine-heme covalent linkage in a hemoglobin. J Am Chem Soc. 2002;124(29):8544-8545. - 30.
Hoy JA, Smagghe BJ, Halder P, Hargrove MS. Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation. Protein Science. 2007;16(2):250-260. - 31.
Nye DB, Lecomte JTJ. Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin. Biochemistry. 2018;57(40):5785-5796. - 32.
Lecomte JTJ, Scott NL, Vu BC, Falzone CJ. Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803. Biochemistry. 2001;40(21):6541-6552. - 33.
Uppal S, Khan MA, Kundu S. Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues. Protein and Peptide Letters. 2020. - 34.
Samuni U, Dantsker D, Ray A, Wittenberg JB, Wittenberg BA, Dewilde S, et al. Kinetic Modulation in Carbonmonoxy Derivatives of Truncated Hemoglobins THE ROLE OF DISTAL HEME POCKET RESIDUES AND EXTENDED APOLAR TUNNEL. Journal of Biological Chemistry. 2003;278(29):27241-27250. - 35.
Hoy JA, Smagghe BJ, Halder P, Hargrove MS. Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation. Protein Sci. 2007;16(2):250-260. - 36.
Thorsteinsson MV, Bevan DR, Potts M, Dou Y, Eich RF, Hargrove MS, et al. A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties. Biochemistry. 1999;38(7):2117-2126. - 37.
Hvitved AN, Trent III JT, Premer SA, Hargrove MS. Ligand binding and hexacoordination in synechocystishemoglobin. Journal of Biological Chemistry. 2001;276(37):34714-34721. - 38.
Goodman MD, Hargrove MS. Quaternary structure of rice nonsymbiotic hemoglobin. Journal of Biological Chemistry. 2001;276(9):6834-6839. - 39.
Hargrove MS. A flash photolysis method to characterize hexacoordinate hemoglobin kinetics. Biophysical Journal. 2000;79(5):2733-2738. - 40.
Hargrove MS, Barry JK, Brucker EA, Berry MB, Phillips Jr GN, Olson JS, et al. Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. Journal of molecular biology. 1997;266(5):1032-1042. - 41.
Olson JS, Mathews AJ, Rohlfs RJ, Springer BA, Egeberg KD, Sligar SG, et al. The role of the distal histidine in myoglobin and haemoglobin. Nature. 1988;336(6196):265-266. - 42.
Rohlfs RJ, Olson JS, Gibson QH. A comparison of the geminate recombination kinetics of several monomeric heme proteins. Journal of Biological Chemistry. 1988;263(4):1803-1813. - 43.
Halder P, Trent Iii JT, Hargrove MS. Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins. PROTEINS: Structure, Function, and Bioinformatics. 2007;66(1):172-182. - 44.
Vinogradov SN, Moens L. Diversity of globin function: enzymatic, transport, storage, and sensing. Journal of Biological Chemistry. 2008;283(14):8773-8777. - 45.
Kundu S, Trent Iii JT, Hargrove MS. Plants, humans and hemoglobins. Trends in plant science. 2003;8(8):387-393. - 46.
Olson JS, Phillips Jr GN. Myoglobin discriminates between O 2, NO, and CO by electrostatic interactions with the bound ligand. JBIC Journal of Biological Inorganic Chemistry. 1997;2(4):544-552. - 47.
Kawada N, Kristensen DB, Asahina K, Nakatani K, Minamiyama Y, Seki S, et al. Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells. Journal of Biological Chemistry. 2001;276(27):25318-25323. - 48.
Sakamoto A, Sakurao S-h, Fukunaga K, Matsubara T, Ueda-Hashimoto M, Tsukamoto S, et al. Three distinct Arabidopsis hemoglobins exhibit peroxidase-like activity and differentially mediate nitrite-dependent protein nitration. FEBS letters. 2004;572(1-3):27-32. - 49.
Gupta KJ, Fernie AR, Kaiser WM, van Dongen JT. On the origins of nitric oxide. Trends in plant science. 2011;16(3):160-168. - 50.
Scott NL, Xu Y, Shen G, Vuletich DA, Falzone CJ, Li Z, et al. Functional and structural characterization of the 2/2 hemoglobin from Synechococcus sp. PCC 7002. Biochemistry. 2010;49(33):7000-7011. - 51.
Uppal S, Khan MA, Kundu S. Identification and characterization of a recombinant cognate hemoglobin reductase from Synechocystis sp. PCC 6803. International Journal of Biological Macromolecules. 2020;162:1054-1063. - 52.
Winslow RM. Blood substitutes--a moving target. Nature medicine. 1995;1(11):1212-1215. - 53.
Winslow RM. Current status of oxygen carriers ('blood substitutes'): 2006. Vox sanguinis. 2006;91(2):102-110. - 54.
Alayash AI. Hemoglobin-based blood substitutes and the treatment of sickle cell disease: more harm than help? Biomolecules. 2017;7(1):2. - 55.
Varadarajan R, Szabo A, Boxer SG. Cloning, expression in Escherichia coli, and reconstitution of human myoglobin. Proceedings of the National Academy of Sciences. 1985;82(17):5681-5684. - 56.
Springer BA, Sligar SG. High-level expression of sperm whale myoglobin in Escherichia coli. Proceedings of the National Academy of Sciences. 1987;84(24):8961-8965. - 57.
Hoffman SJ, Looker DL, Roehrich JM, Cozart PE, Durfee SL, Tedesco JL, et al. Expression of fully functional tetrameric human hemoglobin in Escherichia coli. Proceedings of the National Academy of Sciences. 1990;87(21):8521-8525. - 58.
Looker D, Abbott-Brown D, Cozart P, Durfee S, Hoffman S, Mathews AJ, et al. A human recombinant haemoglobin designed for use as a blood substitute. Nature. 1992;356(6366):258-260. - 59.
Looker D, Mathews AJ, Neway JO, Stetler GL. Expression of recombinant human hemoglobin in Escherichia coli . Methods in enzymology. 231: Elsevier; 1994. p. 364-74. - 60.
Varnado CL, Mollan TL, Birukou I, Smith BJZ, Henderson DP, Olson JS. Development of recombinant hemoglobin-based oxygen carriers. Antioxidants & redox signaling. 2013;18(17):2314-2328. - 61.
Olson JS, Eich RF, Smith LP, Warren JJ, Knowles BC. Protein engineering strategies for designing more stable hemoglobin-based blood substitutes. Artificial Cells, Blood Substitutes, and Biotechnology. 1997;25(1-2):227-241. - 62.
Dou Y, Maillett DH, Eich RF, Olson JS. Myoglobin as a model system for designing heme protein based blood substitutes. Biophysical chemistry. 2002;98(1-2):127-148. - 63.
Ki MR, Pack SP. Fusion tags to enhance heterologous protein expression. Appl Microbiol Biotechnol. 2020;104(6):2411-2425. - 64.
Kosobokova EN, Skrypnik KA, Kosorukov VS. Overview of Fusion Tags for Recombinant Proteins. Biochemistry (Mosc). 2016;81(3):187-200. - 65.
Robert D. Finn, Iouri Kapelioukh, Paine MJI. Rainbow tags: a visual tag system for recombinant protein expression and purification. Biotechniques. 2005;38(3):387-392. - 66.
Kwon SY, Choi YJ, Kang TH, Lee KH, Cha SS, Kim GH, et al. Highly efficient protein expression and purification using bacterial hemoglobin fusion vector. Plasmid. 2005;53(3):274-282. - 67.
Kumar A, Uppal S, Kundu S. The red goldmine: promises of biotechnological riches. 2009. In: Biotechnology Applications [Internet]. I. K. Publishing House, New Delhi, India [230-62].