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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues

Author(s): Sheetal Uppal, Mohd. Asim Khan and Suman Kundu*

Volume 28, Issue 2, 2021

Published on: 13 June, 2020

Page: [164 - 182] Pages: 19

DOI: 10.2174/0929866527666200613220245

Price: $65

Abstract

Aims: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far.

Background: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as “Synechocystis hemoglobin (SynHb)”. The “three histidines” linkages to heme are novel to this cyanobacterial hemoglobin.

Objective: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb.

Methods: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools.

Results: The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a “molten globule” like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability.

Conclusion: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date.

Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

Keywords: hexacoordinate truncated hemoglobin, stability, folding, synechocystis hemoglobin, site-directed mutagenesis, molten globule.

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[1]
Arredondo-Peter, R.; Hargrove, M.S.; Moran, J.F.; Sarath, G.; Klucas, R.V. Plant hemoglobins. Plant Physiol., 1998, 118(4), 1121-1125.
[http://dx.doi.org/10.1104/pp.118.4.1121] [PMID: 9847086]
[2]
Hardison, R. Hemoglobins from bacteria to man: evolution of different patterns of gene expression. J. Exp. Biol., 1998, 201(Pt 8), 1099-1117.
[PMID: 9510523]
[3]
Vinogradov, S.N.; Tinajero-Trejo, M.; Poole, R.K.; Hoogewijs, D. Bacterial and archaeal globins - a revised perspective. Biochim. Biophys. Acta, 2013, 1834(9), 1789-1800.
[http://dx.doi.org/10.1016/j.bbapap.2013.03.021] [PMID: 23541529]
[4]
Wittenberg, J.B.; Bolognesi, M.; Wittenberg, B.A.; Guertin, M. Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J. Biol. Chem., 2002, 277(2), 871-874.
[http://dx.doi.org/10.1074/jbc.R100058200] [PMID: 11696555]
[5]
Pesce, A.; Couture, M.; Dewilde, S.; Guertin, M.; Yamauchi, K.; Ascenzi, P.; Moens, L.; Bolognesi, M. A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family. EMBO J., 2000, 19(11), 2424-2434.
[http://dx.doi.org/10.1093/emboj/19.11.2424] [PMID: 10835341]
[6]
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, 45-64.
[7]
Pesce, A.; Nardini, M.; Milani, M.; Bolognesi, M. Protein structure in the truncated (2/2) hemoglobin family. IUBMB Life, 2007, 59(8-9), 535-541.
[http://dx.doi.org/10.1080/15216540701225933] [PMID: 17701548]
[8]
Vinogradov, S.N.; Hoogewijs, D.; Bailly, X.; Arredondo-Peter, R.; Gough, J.; Dewilde, S.; Moens, L.; Vanfleteren, J.R. A phylogenomic profile of globins. BMC Evol. Biol., 2006, 6, 31.
[http://dx.doi.org/10.1186/1471-2148-6-31] [PMID: 16600051]
[9]
Vinogradov, S.N.; Hoogewijs, D.; Bailly, X.; Arredondo-Peter, R.; Guertin, M.; Gough, J.; Dewilde, S.; Moens, L.; Vanfleteren, J.R. Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life. Proc. Natl. Acad. Sci. USA, 2005, 102(32), 11385-11389.
[http://dx.doi.org/10.1073/pnas.0502103102] [PMID: 16061809]
[10]
Vinogradov, S.N.; Hoogewijs, D.; Bailly, X.; Mizuguchi, K.; Dewilde, S.; Moens, L.; Vanfleteren, J.R. A model of globin evolution. Gene, 2007, 398(1-2), 132-142.
[http://dx.doi.org/10.1016/j.gene.2007.02.041] [PMID: 17540514]
[11]
Hoy, J.A.; Kundu, S.; Trent, J.T., III; Ramaswamy, S.; Hargrove, M.S. The crystal structure of Synechocystis hemoglobin with a covalent heme linkage. J. Biol. Chem., 2004, 279(16), 16535-16542.
[http://dx.doi.org/10.1074/jbc.M313707200] [PMID: 14736872]
[12]
Hoy, J.A.; Smagghe, B.J.; Halder, P.; Hargrove, M.S. Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation. Protein Sci., 2007, 16(2), 250-260.
[http://dx.doi.org/10.1110/ps.062572607] [PMID: 17242429]
[13]
Lecomte, J.T.; Scott, N.L.; Vu, B.C.; Falzone, C.J. Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803. Biochemistry, 2001, 40(21), 6541-6552.
[http://dx.doi.org/10.1021/bi010226u] [PMID: 11371218]
[14]
Hvitved, A.N.; Trent, J.T., III; Premer, S.A.; Hargrove, M.S. Ligand binding and hexacoordination in synechocystis hemoglobin. J. Biol. Chem., 2001, 276(37), 34714-34721.
[http://dx.doi.org/10.1074/jbc.M105175200] [PMID: 11438545]
[15]
Uppal, S.; Salhotra, S.; Mukhi, N.; Zaidi, F.K.; Seal, M.; Dey, S.G.; Bhat, R.; Kundu, S. Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin. J. Biol. Chem., 2015, 290(4), 1979-1993.
[http://dx.doi.org/10.1074/jbc.M114.603225] [PMID: 25451928]
[16]
Cui, Q.; Karplus, M. Allostery and cooperativity revisited. Protein Sci., 2008, 17(8), 1295-1307.
[http://dx.doi.org/10.1110/ps.03259908] [PMID: 18560010]
[17]
Structural Biochemistry/Myoglobin. Available from:. https://en.wikibooks.org/wiki/Structural_Biochemistry/Myoglobin
[18]
Kendrew, J.C.; Bodo, G.; Dintzis, H.M.; Parrish, R.G.; Wyckoff, H.; Phillips, D.C. A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature, 1958, 181(4610), 662-666.
[http://dx.doi.org/10.1038/181662a0] [PMID: 13517261]
[19]
Nishimura, C.; Dyson, H.J.; Wright, P.E. The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate. J. Mol. Biol., 2002, 322(3), 483-489.
[http://dx.doi.org/10.1016/S0022-2836(02)00810-0] [PMID: 12225742]
[20]
Hargrove, M.S.; Krzywda, S.; Wilkinson, A.J.; Dou, Y.; Ikeda-Saito, M.; Olson, J.S. Stability of myoglobin: a model for the folding of heme proteins. Biochemistry, 1994, 33(39), 11767-11775.
[http://dx.doi.org/10.1021/bi00205a012] [PMID: 7918393]
[21]
Olson, J.S. Genetic engineering of myoglobin as a simple prototype for hemoglobin-based blood substitutes. Artif. Cells Blood Substit. Immobil. Biotechnol., 1994, 22(3), 429-441.
[http://dx.doi.org/10.3109/10731199409117872] [PMID: 7994366]
[22]
Olson, J.S.; Mathews, A.J.; Rohlfs, R.J.; Springer, B.A.; Egeberg, K.D.; Sligar, S.G.; Tame, J.; Renaud, J.P.; Nagai, K. The role of the distal histidine in myoglobin and haemoglobin. Nature, 1988, 336(6196), 265-266.
[http://dx.doi.org/10.1038/336265a0] [PMID: 3057383]
[23]
Olson, J.S.; Phillips, G.N.Jr. Kinetic Pathways and Barriers for Ligand Binding to Myoglobin. J. Biol. Chem., 1996, 271(30), 17596.
[http://dx.doi.org/10.1074/jbc.271.30.17593] [PMID: 8663556]
[24]
Olson, J.S.; Phillips, G.N. Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand. J. Biol. Inorg. Chem., 1997, 2, 544-552.
[http://dx.doi.org/10.1007/s007750050169]
[25]
Olson, J.S.; Soman, J.; Phillips, G.N., Jr Ligand pathways in myoglobin: a review of Trp cavity mutations. IUBMB Life, 2007, 59(8-9), 552-562.
[http://dx.doi.org/10.1080/15216540701230495] [PMID: 17701550]
[26]
Culbertson, D.S.; Olson, J.S. Role of heme in the unfolding and assembly of myoglobin. Biochemistry, 2010, 49(29), 6052-6063.
[http://dx.doi.org/10.1021/bi1006942] [PMID: 20540498]
[27]
Hargrove, M.S.; Wilkinson, A.J.; Olson, J.S. Structural factors governing hemin dissociation from metmyoglobin. Biochemistry, 1996, 35(35), 11300-11309.
[http://dx.doi.org/10.1021/bi960372d] [PMID: 8784184]
[28]
Scott, N.L.; Lecomte, J.T. Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the Cyanobacterium synechocystis sp. PCC 6803. Protein Sci., 2000, 9(3), 587-597.
[http://dx.doi.org/10.1110/ps.9.3.587] [PMID: 10752621]
[29]
Semisotnov, G.V.; Rodionova, N.A.; Razgulyaev, O.I.; Uversky, V.N.; Gripas’, A.F.; Gilmanshin, R.I. Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers, 1991, 31(1), 119-128.
[http://dx.doi.org/10.1002/bip.360310111] [PMID: 2025683]
[30]
Santoro, M.M.; Bolen, D.W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry, 1988, 27(21), 8063-8068.
[http://dx.doi.org/10.1021/bi00421a014] [PMID: 3233195]
[31]
Ahmad, F.; Yadav, S.; Taneja, S. Determining stability of proteins from guanidinium chloride transition curves. Biochem. J., 1992, 287(Pt 2), 481-485.
[http://dx.doi.org/10.1042/bj2870481] [PMID: 1445206]
[32]
Jamal, S.; Poddar, N.K.; Singh, L.R.; Dar, T.A.; Rishi, V.; Ahmad, F. Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes. FEBS J., 2009, 276(20), 6024-6032.
[http://dx.doi.org/10.1111/j.1742-4658.2009.07317.x] [PMID: 19765077]
[33]
Swint, L.; Robertson, A.D. Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation. Protein Sci., 1993, 2(12), 2037-2049.
[http://dx.doi.org/10.1002/pro.5560021205] [PMID: 8298454]
[34]
Kumar, A.; Uppal, S.; Kundu, S. The Red Goldmine: Promises of Biotechnological Riches, I., 2008,
[35]
Decatur, S.M.; Belcher, K.L.; Rickert, P.K.; Franzen, S.; Boxer, S.G. Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant. Biochemistry, 1999, 38(34), 11086-11092.
[http://dx.doi.org/10.1021/bi9908888] [PMID: 10460164]
[36]
Kundu, S.; Snyder, B.; Das, K.; Chowdhury, P.; Park, J.; Petrich, J.W.; Hargrove, M.S. The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme. Proteins, 2002, 46(3), 268-277.
[http://dx.doi.org/10.1002/prot.10048] [PMID: 11835502]
[37]
Roach, M.P.; Puspita, W.J.; Watanabe, Y. Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands. J. Inorg. Biochem., 2000, 81(3), 173-182.
[http://dx.doi.org/10.1016/S0162-0134(00)00101-X] [PMID: 11051562]
[38]
Shiro, Y.; Iizuka, T.; Marubayashi, K.; Ogura, T.; Kitagawa, T.; Balasubramanian, S.; Boxer, S.G. Spectroscopic study of Ser92 mutants of human myoglobin: hydrogen bonding effect of Ser92 to proximal His93 on structure and property of myoglobin. Biochemistry, 1994, 33(50), 14986-14992.
[http://dx.doi.org/10.1021/bi00254a006] [PMID: 7999755]
[39]
Smerdon, S.J.; Krzywda, S.; Wilkinson, A.J.; Brantley, R.E., Jr; Carver, T.E.; Hargrove, M.S.; Olson, J.S. Serine92 (F7) contributes to the control of heme reactivity and stability in myoglobin. Biochemistry, 1993, 32(19), 5132-5138.
[http://dx.doi.org/10.1021/bi00070a023] [PMID: 8494890]
[40]
Knappenberger, J.A.; Kuriakose, S.A.; Vu, B.C.; Nothnagel, H.J.; Vuletich, D.A.; Lecomte, J.T. Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin. Biochemistry, 2006, 45(38), 11401-11413.
[http://dx.doi.org/10.1021/bi060691x] [PMID: 16981700]
[41]
Nothnagel, H.J.; Love, N.; Lecomte, J.T. The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin. J. Inorg. Biochem., 2009, 103(1), 107-116.
[http://dx.doi.org/10.1016/j.jinorgbio.2008.09.009] [PMID: 18992944]
[42]
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. EMBO J., 2001, 20(15), 3902-3909.
[http://dx.doi.org/10.1093/emboj/20.15.3902] [PMID: 11483493]
[43]
Trent, J.T., III; Kundu, S.; Hoy, J.A.; Hargrove, M.S. Crystallographic analysis of synechocystis cyanoglobin reveals the structural changes accompanying ligand binding in a hexacoordinate hemoglobin. J. Mol. Biol., 2004, 341(4), 1097-1108.
[http://dx.doi.org/10.1016/j.jmb.2004.05.070] [PMID: 15289104]
[44]
Vu, B.C.; Jones, A.D.; Lecomte, J.T. Novel histidine-heme covalent linkage in a hemoglobin. J. Am. Chem. Soc., 2002, 124(29), 8544-8545.
[http://dx.doi.org/10.1021/ja026569c] [PMID: 12121092]
[45]
Burmester, T.; Ebner, B.; Weich, B.; Hankeln, T. Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Mol. Biol. Evol., 2002, 19(4), 416-421.
[http://dx.doi.org/10.1093/oxfordjournals.molbev.a004096] [PMID: 11919282]
[46]
Dewilde, S.; Kiger, L.; Burmester, T.; Hankeln, T.; Baudin-Creuza, V.; Aerts, T.; Marden, M.C.; Caubergs, R.; Moens, L. Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family. J. Biol. Chem., 2001, 276(42), 38949-38955.
[http://dx.doi.org/10.1074/jbc.M106438200] [PMID: 11473128]
[47]
Goodman, M.D.; Hargrove, M.S. Quaternary structure of rice nonsymbiotic hemoglobin. J. Biol. Chem., 2001, 276(9), 6834-6839.
[http://dx.doi.org/10.1074/jbc.M009254200] [PMID: 11106662]
[48]
Mukhi, N.; Dhindwal, S.; Uppal, S.; Kumar, P.; Kaur, J.; Kundu, S. X-ray crystallographic structural characteristics of Arabidopsis hemoglobin I and their functional implications. Biochim. Biophys. Acta, 2013, 1834(9), 1944-1956.
[http://dx.doi.org/10.1016/j.bbapap.2013.02.024] [PMID: 23485912]
[49]
Hargrove, M.S.; Olson, J.S. The stability of holomyoglobin is determined by heme affinity. Biochemistry, 1996, 35(35), 11310-11318.
[http://dx.doi.org/10.1021/bi9603736] [PMID: 8784185]
[50]
Banerjee, M.; Pramanik, M.; Bhattacharya, D.; Lahiry, M.; Chakrabarti, A. Faster heme loss from hemoglobin E than HbS, in acidic pH: effect of aminophospholipids. J. Biosci., 2011, 36(5), 809-816.
[http://dx.doi.org/10.1007/s12038-011-9163-5] [PMID: 22116278]
[51]
Akiyama, K.; Fukuda, M.; Kobayashi, N.; Matsuoka, A.; Shikama, K. The pH-dependent swinging-out of the distal histidine residue in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi). Biochim. Biophys. Acta, 1994, 1208(2), 306-309.
[http://dx.doi.org/10.1016/0167-4838(94)90117-1] [PMID: 7947962]
[52]
Uppal, S.; Kumar, A.; Shandilya, M.; Mukhi, N.; Singh, A.K.; Kateriya, S.; Kaur, J.; Kundu, S. Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts. Anal. Biochem., 2016, 510, 120-128.
[http://dx.doi.org/10.1016/j.ab.2016.07.014] [PMID: 27449132]
[53]
Uversky, V.N.; Semisotnov, G.V.; Pain, R.H.; Ptitsyn, O.B. ‘All-or-none’ mechanism of the molten globule unfolding. FEBS Lett., 1992, 314(1), 89-92.
[http://dx.doi.org/10.1016/0014-5793(92)81468-2] [PMID: 1451808]
[54]
Ptitsyn, O.B. Molten globule and protein folding. Adv. Protein Chem., 1995, 47, 83-229.
[http://dx.doi.org/10.1016/S0065-3233(08)60546-X] [PMID: 8561052]
[55]
Ferrer, M.; Barany, G.; Woodward, C. Partially folded, molten globule and molten coil states of bovine pancreatic trypsin inhibitor. Nat. Struct. Biol., 1995, 2(3), 211-217.
[http://dx.doi.org/10.1038/nsb0395-211] [PMID: 7539710]
[56]
Kitto, G.B.; Thomas, P.W.; Hackert, M.L. Evolution of cooperativity in hemoglobins: what can invertebrate hemoglobins tell us? J. Exp. Zool., 1998, 282(1-2), 120-126.
[http://dx.doi.org/10.1002/(SICI)1097-010X(199809/10)282:1/2<120::AID-JEZ13>3.0.CO;2-Y] [PMID: 9723169]
[57]
Perrella, M.; Davids, N.; Rossi-Bernardi, L. The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates. Implications on the mechanism of cooperativity. J. Biol. Chem., 1992, 267(13), 8744-8751.
[PMID: 1577717]
[58]
Huestis, W.H.; Raftery, M.A. Conformation and cooperativity in hemoglobin. Biochemistry, 1975, 14(9), 1886-1892.
[http://dx.doi.org/10.1021/bi00680a013] [PMID: 235969]
[59]
Szabo, A.; Karplus, M. Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions. Biochemistry, 1975, 14(5), 931-940.
[http://dx.doi.org/10.1021/bi00676a009] [PMID: 235946]
[60]
Christensen, H.; Pain, R.H. Molten globule intermediates and protein folding. Eur. Biophys. J., 1991, 19(5), 221-229.
[http://dx.doi.org/10.1007/BF00183530] [PMID: 2060495]

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