Truncated hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002: evidence for hexacoordination and covalent adduct formation in the ferric recombinant protein.
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.
Scott
Nancy L
NL
Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Falzone
Christopher J
CJ
Vuletich
David A
DA
Zhao
Jindong
J
Bryant
Donald A
DA
Lecomte
Juliette T J
JT
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D000458:Cyanobacteria / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011524:Protoporphyrins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D054793:Truncated Hemoglobins
D006454:Hemoglobins; D011524:Protoporphyrins; D011994:Recombinant Proteins; D054793:Truncated Hemoglobins; C448299:iron protoporphyrin IX; D006639:Histidine