Cyanide Binding to Hexacoordinate Cyanobacterial Hemoglobins:  Hydrogen-Bonding Network and Heme Pocket Rearrangement in Ferric H117A Synechocystis Hemoglobin

Abstract

The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of 1H2O/2H2O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the 15N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.

Slow Ligand Binding Kinetics Dominate Ferrous Hexacoordinate Hemoglobin Reactivities and Reveal Differences between Plants and Other Species

“…The crystal structures of SynHb in the presence and absence of bound ligand support this possibility (29). When an exogenous ligand is bound, His 46 (which coordinates the ligand binding site in the absence of exogenous ligands) moves completely out of the heme pocket and is accompanied by major rearrangements in the B and E helices and the EF loop (30). It is possible that the conformational change that facilitates hexacoordination following flash photolysis follows more than one pathway, causing heterogeneity in the time course for ligand rebinding.…”

Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

“…This difference increased to 701 ppm in the A69S variant. Evidence for Hbonding of the bound cyanide is provided by the 22-ppm exchangeable signal attributed to Tyr22 (B10) Oη 1 H in Figure 3C (37). Thus, this aspect of the structure appeared conserved.…”

“…The proximal-side substitution H117A has the remarkable consequence of sharpening the heme resonances (37). The A69S replacement did not have the same effect ( Figure 3C); the lines for the cyanide complex remained as broad as for the wild-type, but the shifts were affected to some extent.…”

“…At 32 °C and neutral pH, the difference in chemical shift between free and bound cyanide was determined to be 692 ppm in the wildtype protein (37). This difference increased to 701 ppm in the A69S variant.…”

“…An approximate pseudo-first-order rate constant was obtained for the phase that accounted for the highest fraction of the population, typically > 70%. The values at the three cyanide concentrations were used to obtain an apparent bimolecular rate constant as in previous work on the wild-type and H117A rHb-Rs (37). All measurements were performed in triplicate at pH 7.2 (100 mM phosphate) and were repeated in D 2 O (pH 7.2, uncorrected for isotope effect).…”