Truncated Hemoglobin from the Cyanobacterium Synechococcus sp. PCC 7002:  Evidence for Hexacoordination and Covalent Adduct Formation in the Ferric Recombinant Protein^,

Abstract

The glbN gene for the hemoglobin of Synechoccocus sp. PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli. The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein. Mass spectrometric analysis confirmed the identity of the polypeptide. NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp. PCC 6803 [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side. Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix. In this protein, as in that from Synechocystis sp. PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein. Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

“…Alternatively, the multiple hexacoordinated states may account for multiple functions in the same molecule. Hexacoordinated Hbs are observed in unicellular eukaryotes [17], plants [49], invertebrates [50], and in some tissues of higher vertebrates [48,51], but only a few cases have been examined and reported in the literature for bacterial 2/2 Hbs [52][53][54][55]. The physiological role of these hexacoordinated Hbs is not well understood.…”

Protein structure in the truncated (2/2) hemoglobin family

“…As mentioned above, Ss-2/2HbN displays bis-histidine heme hexacoordination, where HisF8 and HisE10 are the Fe-atom ligands (21,22,32). Binding of an exogenous ligand to the heme distal site, requires the dissociation of HisE10 from the heme and remarkable conformational changes affecting the B-and E-helices (12,33).…”

The Crystal Structure of Synechocystis Hemoglobin with a Covalent Heme Linkage

“…The ferric protein used for crystallization was produced under conditions similar to those in which the His 117 -heme covalent link was observed by NMR experiments in SynHb and the homologue in Synechococcus (28,29). However, the reported method of dithionite treatment leading to the covalent link (27,28) was not used in our treatment of the protein.…”