Truncated hemoglobins (trHbs) are heme proteins found in bacteria, plants, and unicellular eukaryotes. They are distantly related to vertebrate hemoglobins and are typically shorter than these by 20-40 residues. The multiple amino acid deletions, insertions, and replacements result in distinctive alterations of the canonical globin fold and a wide range of chemical properties. An early phylogenetic analysis categorized trHbs into three groups, I (trHbN), II (trHbO), and III (trHbP). Here, we revisit this analysis with 111 trHbs. We find that trHbs are orthologous within each group and paralogous across the groups. Group I globins form the most disparate set and separate into two divergent subgroups. Group II is comparatively homogeneous, whereas Group III displays the highest level of overall conservation. In Group I and Group II globins, for which some ligand binding and structural data are available, an improved description of probable protein-ligand interactions is achieved. Other conservation trends are either confirmed (essential glycines in loops), refined (lining of ligand access tunnel), or newly identified (helix start signal). The Group III globins, so far uncharacterized, exhibit recognizable heme cavity residues while lacking some of the residues thought to be important to the trHb fold. An analysis of the phylogenetic trees of each group provides a plausible scenario for the emergence of trHbs, by which the Group II trHb gene was the original gene, and the Group I trHb and Group III trHb genes were obtained via duplication and transfer events.

The glbN gene for the hemoglobin of Synechoccocus sp. PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli. The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein. Mass spectrometric analysis confirmed the identity of the polypeptide. NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp. PCC 6803 [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side. Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix. In this protein, as in that from Synechocystis sp. PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein. Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

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.

Cyanobacterium Synechococcus sp. PCC 7002 contains a single gene (glbN) coding for GlbN, a protein of the 2/2 hemoglobin lineage. The precise function of GlbN is not known, but comparison to similar 2/2 hemoglobins suggests that reversible dioxygen binding is not its main activity. In this report, the results of in vitro and in vivo experiments probing the role of GlbN are presented. Transcription profiling indicated that glbN is not strongly regulated under any of a large number of growth conditions and that the gene is probably constitutively expressed. High levels of nitrate, used as the sole source of nitrogen, and exposure to nitric oxide were tolerated better by the wild-type strain than a glbN null mutant, whereas overproduction of GlbN in the null mutant background restored the wild-type growth. The cellular contents of reactive oxygen/nitrogen species were elevated in the null mutant under all conditions and were highest under NO challenge or in the presence of high nitrate concentrations. GlbN overproduction attenuated these contents significantly under the latter conditions. The analysis of cell extracts revealed that the heme of GlbN was covalently bound to overproduced GlbN apoprotein in cells grown under microoxic conditions. A peroxidase assay showed that purified GlbN does not possess significant hydrogen peroxidase activity. It was concluded that GlbN protects cells from reactive nitrogen species that could be encountered naturally during growth on nitrate or under denitrifying conditions. The solution structure of covalently modified GlbN was determined and used to rationalize some of its chemical properties.

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