Structural characterization of the truncated hemoglobin from Synechocystis sp. PCC 6803

This thesis details studies of the heme pocket structure from the truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803. Synechocystis sp. PCC 6803 Hb contains an endogenously hexacoordinate heme iron, with His46 (distal) and His70 (proximal) serving as axial ligands. The solution structure of the ferric form (hemichrome), revealed a highly alpha-helical protein with a fold similar to that of other truncated hemoglobins [Falzone, C. J. et al. (2002) J. Mol. Biol. 324, 1015]. Characterization of the protein in solution showed that it is able to undergo a posttranslational modification of the heme 2-vinyl group [Scott, N. L. et al. (2002) Biochemistry 41, 6902]. The reaction was facilitated by sodium dithionite reduction of the iron. Optical and NMR data indicated that the heme iron in the product remained low-spin Fe(III). Mass spectrometry and NMR spectroscopy established that a covalent link was formed between His117 and the alpha-position of the 2-vinyl group. Optical methods showed that covalent attachment of the heme group stabilized the protein by >20°C with respect to thermal denaturation and 1.1 pH units with respect to acid denaturation. The unique reactivity of His117 was confirmed by mutagenesis. Thus His117Ala Synechocystis sp. PCC 6803 Hb was incapable of forming the cross-link. Cyanide binding experiments were performed on this mutant to characterize the metcyano form in the absence of the cross-link. It was found that His46 (distal) was displaced from the heme iron. A hydrogen bonding network involving the cyanide ion, Tyr22, Gln43 and Gln47 was detected, likely mimicking that in the related hemoglobins from Chlamydomonas eugametos and Mycobacterium tuberculosis. Kinetic data indicated that the variant bound cyanide more slowly than the wild-type protein and suggested that position 117 had an influence on the properties of the heme pocket. Ligand binding in hemoglobins is known to be modulated by structural interactions at the proximal and distal ligand sites. Here, hyperfine chemical shifts were used to probe the relationship between heme electronic structure and axial ligand orientation. The data indicated that the covalent modification perturbed moderately the orientation of the axial ligands and overall heme electronic structure. The heme pocket structure in the ligand dissociated state was probed by incorporation of Zn(II) protoporphyrin IX in place of heme. NMR studies on the pentacoordinate zinc protein revealed that His46 moved away from the porphyrin ring; however, the conformation was neither that observed in the hemichrome, nor that in the metcyano complex. The hemoglobin characterization presented here demonstrated new reactivity and conformational properties expanding in unexpected ways the structure-function relationship within this family of proteins.