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 μM.
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.