Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803.
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.
Scott
N L
NL
Department of Chemistry and the Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
Lecomte
J T
JT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D013053:Spectrophotometry
D001426:Bacterial Proteins; D006454:Hemoglobins; D008024:Ligands; C407880:slr2097 protein, Synechocystis; D006418:Heme