Characterization of the heme–histidine cross-link in cyanobacterial hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002

Abstract

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

“…The backbone superposition of the ferric structures in Figure 1A shows minimal backbone deviation (RMSD ¼ 0.45 Å ), but small changes in the orientation of the heme and axial histidines, in agreement with the partial structural observations obtained by NMR (Vu et al 2004b). The superposition in Figure 1B shows that, relative to the heme, mutation of H117 causes outward movement of the H-helix with minor movements of the F-and G-helices (RMSD ¼ 1.08 Å ).…”

“…However, the experiments presented here do not distinguish between these contributions. Vu et al (2004b) report that decoordination of both axial histidines accompanies protein unfolding in both ferric wt SynHb forms (with and without the covalent bond) due to the presence of isosbestic points in the spectral transitions. However, their ferric, unfolded spectra are similar to those reported here, including the difference seen with and without the covalent bond.…”

“…These experiments included observation of slow heme release (Scott and Lecomte 2000;Lecomte et al 2001), thermal denaturation (Lecomte et al 2001), and measurement of the electrochemical midpoint potential (Lecomte et al 2001). After the covalent bond was discovered as the source of heterogeneity, partial NMR structural analysis indicated little global conformational change among the ferric tertiary structures of the two forms of wild-type protein and the H117A mutant protein (which is incapable of forming the covalent bond) (Falzone et al 2002;Vu et al 2004aVu et al , 2004b. Thermal and acid denaturation studies of the ferric forms of these three proteins found the wildtype protein with the covalent bond to be more stable than the mutant or wild-type lacking the bond (Lecomte et al 2001;Vu et al 2004a,b).…”

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

“…SynHb is a hexacoordinated and truncated bacterial Hb that displays two-on-two ␣-helical globin fold and unique features not observed for any other Hb (4,56,57). The present investigation, partly an analysis of the influence of key heme pocket residues on SynHb stability, validated some earlier propositions and provided additional extended insight (7,26).…”

Isolation and characterization of a new phycoerythrin from the cyanobacterium Synechococcus sp. ECS-18

“…The nucleotide sequences of the β-and α-subunits of SCH-phycoerythrin showed strong homologies to those from other Synechococcus spp. including WH8103, WH7803 and RCC307 (de Lorimier et al 1992;Vu et al 2004).…”