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. 2000 Mar;9(3):587–597. doi: 10.1110/ps.9.3.587

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803.

N L Scott 1, J T Lecomte 1
PMCID: PMC2144580  PMID: 10752621

Abstract

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.

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Selected References

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  1. Alam S. L., Satterlee J. D. Complete heme proton hyperfine resonance assignments of the Glycera dibranchiata component IV metcyano monomer hemoglobin. Biochemistry. 1994 Apr 5;33(13):4008–4018. doi: 10.1021/bi00179a030. [DOI] [PubMed] [Google Scholar]
  2. Andrade M. A., Chacón P., Merelo J. J., Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 1993 Jun;6(4):383–390. doi: 10.1093/protein/6.4.383. [DOI] [PubMed] [Google Scholar]
  3. Angeloni S. V., Potts M. Analysis of the sequences within and flanking the cyanoglobin-encoding gene, glbN, of the cyanobacterium Nostoc commune UTEX 584. Gene. 1994 Aug 19;146(1):133–134. doi: 10.1016/0378-1119(94)90848-6. [DOI] [PubMed] [Google Scholar]
  4. Arnold E. V., Bohle D. S., Jordan P. A. Reversible and irreversible hemichrome generation by the oxygenation of nitrosylmyoglobin. Biochemistry. 1999 Apr 13;38(15):4750–4756. doi: 10.1021/bi982729e. [DOI] [PubMed] [Google Scholar]
  5. Arnoux P., Haser R., Izadi N., Lecroisey A., Delepierre M., Wandersman C., Czjzek M. The crystal structure of HasA, a hemophore secreted by Serratia marcescens. Nat Struct Biol. 1999 Jun;6(6):516–520. doi: 10.1038/9281. [DOI] [PubMed] [Google Scholar]
  6. Arredondo-Peter R., Hargrove M. S., Sarath G., Moran J. F., Lohrman J., Olson J. S., Klucas R. V. Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli. Plant Physiol. 1997 Nov;115(3):1259–1266. doi: 10.1104/pp.115.3.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Becker M., Schäfer G. Purification and spectral characterization of a b-type cytochrome from the plasma membrane of the archaebacterium Sulfolobus acidocaldarius. FEBS Lett. 1991 Oct 21;291(2):331–335. doi: 10.1016/0014-5793(91)81314-x. [DOI] [PubMed] [Google Scholar]
  8. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S., Barry C. E., 3rd Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998 Jun 11;393(6685):537–544. doi: 10.1038/31159. [DOI] [PubMed] [Google Scholar]
  9. Couture M., Chamberland H., St-Pierre B., Lafontaine J., Guertin M. Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos. Mol Gen Genet. 1994 Apr;243(2):185–197. doi: 10.1007/BF00280316. [DOI] [PubMed] [Google Scholar]
  10. Couture M., Das T. K., Lee H. C., Peisach J., Rousseau D. L., Wittenberg B. A., Wittenberg J. B., Guertin M. Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands. J Biol Chem. 1999 Mar 12;274(11):6898–6910. doi: 10.1074/jbc.274.11.6898. [DOI] [PubMed] [Google Scholar]
  11. Couture M., Guertin M. Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos. Eur J Biochem. 1996 Dec 15;242(3):779–787. doi: 10.1111/j.1432-1033.1996.0779r.x. [DOI] [PubMed] [Google Scholar]
  12. Couture M., Yeh S. R., Wittenberg B. A., Wittenberg J. B., Ouellet Y., Rousseau D. L., Guertin M. A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11223–11228. doi: 10.1073/pnas.96.20.11223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Das T. K., Couture M., Lee H. C., Peisach J., Rousseau D. L., Wittenberg B. A., Wittenberg J. B., Guertin M. Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme. Biochemistry. 1999 Nov 16;38(46):15360–15368. doi: 10.1021/bi991237e. [DOI] [PubMed] [Google Scholar]
  14. Das T. K., Lee H. C., Duff S. M., Hill R. D., Peisach J., Rousseau D. L., Wittenberg B. A., Wittenberg J. B. The heme environment in barley hemoglobin. J Biol Chem. 1999 Feb 12;274(7):4207–4212. doi: 10.1074/jbc.274.7.4207. [DOI] [PubMed] [Google Scholar]
  15. Duff S. M., Wittenberg J. B., Hill R. D. Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands. J Biol Chem. 1997 Jul 4;272(27):16746–16752. doi: 10.1074/jbc.272.27.16746. [DOI] [PubMed] [Google Scholar]
  16. Emerson S. D., La Mar G. N. NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand. Biochemistry. 1990 Feb 13;29(6):1556–1566. doi: 10.1021/bi00458a029. [DOI] [PubMed] [Google Scholar]
  17. Hanania G. I., Yeghiayan A., Cameron B. F. Absorption spectra of sperm-whale ferrimyoglobin. Biochem J. 1966 Jan;98(1):189–192. doi: 10.1042/bj0980189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hardison R. Hemoglobins from bacteria to man: evolution of different patterns of gene expression. J Exp Biol. 1998 Apr;201(Pt 8):1099–1117. doi: 10.1242/jeb.201.8.1099. [DOI] [PubMed] [Google Scholar]
  19. Hargrove M. S., Wilkinson A. J., Olson J. S. Structural factors governing hemin dissociation from metmyoglobin. Biochemistry. 1996 Sep 3;35(35):11300–11309. doi: 10.1021/bi960372d. [DOI] [PubMed] [Google Scholar]
  20. Hill D. R., Belbin T. J., Thorsteinsson M. V., Bassam D., Brass S., Ernst A., Böger P., Paerl H., Mulligan M. E., Potts M. GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp. J Bacteriol. 1996 Nov;178(22):6587–6598. doi: 10.1128/jb.178.22.6587-6598.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Howitt C. A., Vermaas W. F. Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803. Biochemistry. 1998 Dec 22;37(51):17944–17951. doi: 10.1021/bi981486n. [DOI] [PubMed] [Google Scholar]
  22. Iwaasa H., Takagi T., Shikama K. Protozoan hemoglobin from Tetrahymena pyriformis. Isolation, characterization, and amino acid sequence. J Biol Chem. 1990 May 25;265(15):8603–8609. [PubMed] [Google Scholar]
  23. Iwaasa H., Takagi T., Shikama K. Protozoan myoglobin from Paramecium caudatum. Its unusual amino acid sequence. J Mol Biol. 1989 Jul 20;208(2):355–358. doi: 10.1016/0022-2836(89)90395-1. [DOI] [PubMed] [Google Scholar]
  24. Kaneko T., Sato S., Kotani H., Tanaka A., Asamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996 Jun 30;3(3):109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
  25. Keller R. M., Wüthrich K. Structural study of the heme crevice in cytochrome b5 based on individual assignments of the 1H-NMR lines of the heme group and selected amino acid residues. Biochim Biophys Acta. 1980 Feb 27;621(2):204–217. doi: 10.1016/0005-2795(80)90172-5. [DOI] [PubMed] [Google Scholar]
  26. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  27. Kunst F., Ogasawara N., Moszer I., Albertini A. M., Alloni G., Azevedo V., Bertero M. G., Bessières P., Bolotin A., Borchert S. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997 Nov 20;390(6657):249–256. doi: 10.1038/36786. [DOI] [PubMed] [Google Scholar]
  28. La Mar G. N., Burns P. D., Jackson J. T., Smith K. M., Langry K. C., Strittmatter P. Proton magnetic resonance determination of the relative heme orientations in disordered native and reconstituted ferricytochrome b5. Assignment of heme resonances by deuterium labeling. J Biol Chem. 1981 Jun 25;256(12):6075–6079. [PubMed] [Google Scholar]
  29. La Mar G. N., Davis N. L., Parish D. W., Smith K. M. Heme orientational disorder in reconstituted and native sperm whale myoglobin. Proton nuclear magnetic resonance characterizations by heme methyl deuterium labeling in the Met-cyano protein. J Mol Biol. 1983 Aug 25;168(4):887–896. doi: 10.1016/s0022-2836(83)80080-1. [DOI] [PubMed] [Google Scholar]
  30. Li W. H. Retention of cryptic genes in microbial populations. Mol Biol Evol. 1984 Feb;1(2):213–219. doi: 10.1093/oxfordjournals.molbev.a040312. [DOI] [PubMed] [Google Scholar]
  31. Moens L., Vanfleteren J., Van de Peer Y., Peeters K., Kapp O., Czeluzniak J., Goodman M., Blaxter M., Vinogradov S. Globins in nonvertebrate species: dispersal by horizontal gene transfer and evolution of the structure-function relationships. Mol Biol Evol. 1996 Feb;13(2):324–333. doi: 10.1093/oxfordjournals.molbev.a025592. [DOI] [PubMed] [Google Scholar]
  32. Potts M., Angeloni S. V., Ebel R. E., Bassam D. Myoglobin in a cyanobacterium. Science. 1992 Jun 19;256(5064):1690–1691. doi: 10.1126/science.256.5064.1690. [DOI] [PubMed] [Google Scholar]
  33. Qin J., La Mar G. N., Dou Y., Admiraal S. J., Ikeda-Saito M. 1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant. Coordination of His64(E11) at the sixth position in both low-spin and high-spin states. J Biol Chem. 1994 Jan 14;269(2):1083–1090. [PubMed] [Google Scholar]
  34. Rachmilewitz E. A., Peisach J., Blumberg W. E. Studies on the stability of oxyhemoglobin A and its constituent chains and their derivatives. J Biol Chem. 1971 May 25;246(10):3356–3366. [PubMed] [Google Scholar]
  35. Rance M., Sørensen O. W., Bodenhausen G., Wagner G., Ernst R. R., Wüthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun. 1983 Dec 16;117(2):479–485. doi: 10.1016/0006-291x(83)91225-1. [DOI] [PubMed] [Google Scholar]
  36. Rifkind J. M., Abugo O., Levy A., Heim J. Detection, formation, and relevance of hemichromes and hemochromes. Methods Enzymol. 1994;231:449–480. doi: 10.1016/0076-6879(94)31030-0. [DOI] [PubMed] [Google Scholar]
  37. STRITTMATTER P. The nature of the heme binding in microsomal cytochrome b5. J Biol Chem. 1960 Aug;235:2492–2497. [PubMed] [Google Scholar]
  38. Shikama K., Matsuoka A., Iwaasa H. The unique structures of protozoan myoglobin and yeast hemoglobin: an evolutionary diversity. Int J Biochem Cell Biol. 1995 Nov;27(11):1107–1115. doi: 10.1016/1357-2725(95)00084-3. [DOI] [PubMed] [Google Scholar]
  39. TEALE F. W. Cleavage of the haem-protein link by acid methylethylketone. Biochim Biophys Acta. 1959 Oct;35:543–543. doi: 10.1016/0006-3002(59)90407-x. [DOI] [PubMed] [Google Scholar]
  40. Telford J. R., Tezcan F. A., Gray H. B., Winkler J. R. Role of ligand substitution in ferrocytochrome c folding. Biochemistry. 1999 Feb 9;38(6):1944–1949. doi: 10.1021/bi981933z. [DOI] [PubMed] [Google Scholar]
  41. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thorsteinsson M. V., Bevan D. R., Ebel R. E., Weber R. E., Potts M. Spectroscopical and functional characterization of the hemoglobin of Nostoc commune (UTEX 584 (Cyanobacterial). Biochim Biophys Acta. 1996 Jan 4;1292(1):133–139. doi: 10.1016/0167-4838(95)00178-6. [DOI] [PubMed] [Google Scholar]
  43. Thorsteinsson M. V., Bevan D. R., Potts M., Dou Y., Eich R. F., Hargrove M. S., Gibson Q. H., Olson J. S. A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties. Biochemistry. 1999 Feb 16;38(7):2117–2126. doi: 10.1021/bi9819172. [DOI] [PubMed] [Google Scholar]
  44. Tsubamoto Y., Matsuoka A., Yusa K., Shikama K. Protozoan myoglobin from Paramecium caudatum. Its autoxidation reaction and hemichrome formation. Eur J Biochem. 1990 Oct 5;193(1):55–59. doi: 10.1111/j.1432-1033.1990.tb19303.x. [DOI] [PubMed] [Google Scholar]
  45. Whitaker T. L., Berry M. B., Ho E. L., Hargrove M. S., Phillips G. N., Jr, Komiyama N. H., Nagai K., Olson J. S. The D-helix in myoglobin and in the beta subunit of hemoglobin is required for the retention of heme. Biochemistry. 1995 Jul 4;34(26):8221–8226. doi: 10.1021/bi00026a002. [DOI] [PubMed] [Google Scholar]
  46. Wishart D. S., Bigam C. G., Yao J., Abildgaard F., Dyson H. J., Oldfield E., Markley J. L., Sykes B. D. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR. 1995 Sep;6(2):135–140. doi: 10.1007/BF00211777. [DOI] [PubMed] [Google Scholar]
  47. Wood P. M. Bacterial proteins with CO-binding b- or c-type haem. Functions and absorption spectroscopy. Biochim Biophys Acta. 1984 Dec 17;768(3-4):293–317. doi: 10.1016/0304-4173(84)90020-x. [DOI] [PubMed] [Google Scholar]
  48. Wu J. Z., La Mar G. N., Yu L. P., Lee K. B., Walker F. A., Chiu M. L., Sligar S. G. 1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation. Biochemistry. 1991 Feb 26;30(8):2156–2165. doi: 10.1021/bi00222a020. [DOI] [PubMed] [Google Scholar]
  49. Yamauchi K., Tada H., Usuki I. Structure and evolution of Paramecium hemoglobin genes. Biochim Biophys Acta. 1995 Oct 17;1264(1):53–62. doi: 10.1016/0167-4781(95)00114-v. [DOI] [PubMed] [Google Scholar]
  50. Yeh S. R., Takahashi S., Fan B., Rousseau D. L. Ligand exchange during cytochrome c folding. Nat Struct Biol. 1997 Jan;4(1):51–56. doi: 10.1038/nsb0197-51. [DOI] [PubMed] [Google Scholar]

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