Functional and Structural Characterization of the 2/2 Hemoglobin from Synechococcus sp. PCC 7002,

Abstract

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.

Bacterial and archaeal globins — A revised perspective

“…These include four TrHb1s, Mycobacterium tuberculosis [71], Geobacillus stearothermophilus [72], and the cyanobacteria Synechocystis sp.PCC7002 [73][74] and Synechococcus sp. PCC7002 [75]; three TrHb2s, Mycobacterium tuberculosis [76], Bacillus subtilis [77] and Thermobifida fusca [78] and two TrHb3s, Campylobacter jejuni [79] and Helicobacter hepaticus [80]. The structures provide invaluable information on the choice of residues that can be fruitfully studied by site-directed mutagenesis and are critical, of course for quantum mechanical modeling of ligand access and other sub-molecular properties.…”

Covalent attachment of the heme to Synechococcus hemoglobin alters its reactivity toward nitric oxide

“…Several studies of the transcriptome of Synechococcus cells have detected constitutive expression of glbN [16–18]. When overexpressed under microoxic conditions, the protein contains covalently attached heme whereas oxic conditions appear to favor unmodified GlbN [16]. Thus, both GlbN and GlbN-A are candidates for physiological relevance, and the PTM may act as a sensor for cellular redox and oxygen status.…”

The broad diversity of heme-protein cross-links: An overview

“…6A) and PCC 7002 (PDB ID: 4MAX [66], Fig. 6B), respectively, as well as NMR structure for PCC 7002 (PDB ID: 2KSC [67]). The combination of site-directed mutagenesis, NMR and optical spectroscopic studies provided evidence that the His-heme cross-link in reduced cyanobacteria Hbs was formed by an electrophilic addition with a reduction-driven mechanism (Scheme 4) [68].…”

“…It was shown that introduction of a cysteine at position 117 (H117C mutation) of PCC 6803 can result in formation of a thioether bond with the heme 2-vinyl group [68], suggesting that this position is suitable for both His-heme and Cys-heme cross-links. Despite the fact, cyanobacteria Hbs prefer the His-heme cross-link, likely due to its advantages such as less interference under high oxidative and nitrosative stress conditions that many cyanobacteria encounter [67].…”