In vitro investigations of the hemoglobins from the bacteria Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, and Helicobacter hepaticus

Heme is utilized by many proteins to expand their chemical repertoire. An important aspect of heme protein chemistry is how the polypeptide chain adjusts the reactivity of the cofactor so that it performs different functions. This thesis describes the in vitro characterization of representative bacterial hemoglobins (Hbs) from the 2/2 lineage of the Hb superfamily.;The 2/2 Hbs of the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 exhibit bis-histidyl coordination of the heme iron, but are capable of binding small exogenous ligands by displacement of the distal side histidine. These Hbs also undergo a post-translational modification that affixes the heme to the polypeptide chain by a vinyl---histidine bond. The repercussions of the covalent heme adduct and bis-histidyl ligation were inspected using a variety of techniques including NMR and optical spectroscopies. Both features were found to influence cyanide binding to the ferric state Unlike in some hexacoordinate hemoglobins, distal histidine ligation to the iron was insufficient to prevent H2O2-induced oxidative damage in Synechocystis 6803 Hb. Coordination by the histidine did, however, direct the post-translational modification preferentially to the heme 2-vinyl. Studies were also performed to determine the mechanism of the in vitro formation of the heme-protein linkage. The data were consistent with a reductive route and a carbocation intermediate. No evidence was found for the involvement of dioxygen. Attempts were made to introduce a similar heme-protein bond in rat microsomal cytochrome b5 using site directed mutagenesis to place a histidine in proximity of the heme vinyl substituents. Bond formation was not observed, which suggested that the precise control of stereo-electronic factors was necessary for the reaction and explained why this post-translational modification is rare.;Finally, an expression and purification system was developed for the 2/2 Hb of the proteobacterium Helicobacter hepaticus. Preliminary characterization showed that the protein shares several structural properties with the 2/2 Hb of Campylobacter jejuni and has the ability to form a stable ferrous-cyanide complex.;2/2 Hbs are found in small amounts in bacterial cells and in the large majority of cases, their function is not known. The in vitro studies provided novel information on the chemical properties of these globins and will guide functional studies.