Structural and dynamic repercussions of heme binding and heme-protein cross-linking in Synechococcus sp. PCC 7002 hemoglobin.
The recombinant two-on-two hemoglobin from the cyanobacterium Synechoccocus sp. PCC 7002 (S7002 rHb) is a bishistidine hexacoordinate globin capable of forming a covalent cross-link between a heme vinyl and a histidine in the C-terminal helix (H helix). Of the two heme axial histidines, His46 (in the E helix, distal side) and His70 (in the F helix, proximal histidine), His46 is displaced by exogenous ligands. S7002 rHb can be readily prepared as an apoglobin (apo-rHb), a non-cross-linked hemichrome (ferric iron and histidine axial ligands, rHb-R), and a cross-linked hemichrome (rHb-A). To determine the effects of heme binding and subsequent cross-linking, apo-rHb, rHb-R, and rHb-A were subjected to thermal denaturation and 1H/2H exchange. Interpretation of the latter data was based on nuclear magnetic resonance assignments obtained with uniformly 15N- and 13C,15N-labeled proteins. Apo-rHb was found to contain a cooperative structural core, which was extended and stabilized by heme binding. Cross-linking resulted in further stabilization attributed mainly to an unfolded-state effect. Protection factors were higher at the cross-link site and near His70 in rHb-A than in rHb-R. In contrast, other regions became less resistant to exchange in rHb-A. These included portions of the B and E helices, which undergo large conformational changes upon exogenous ligand binding. Thus, the cross-link readjusted the dynamic properties of the heme pocket. 1H/2H exchange data also revealed that the B, G, and H helices formed a robust core regardless of the presence of the heme or cross-link. This motif likely encompasses the early folding nucleus of two-on-two globins.
Vuletich
David A
DA
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Falzone
Christopher J
CJ
Lecomte
Juliette T J
JT
D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D046940:Synechococcus / Q000378:metabolism*
D006454:Hemoglobins; D008024:Ligands; D006418:Heme