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

• Published in: Journal of inorganic biochemistry 2017-12, Vol.177, p.171-182
• Main Authors: Preimesberger, Matthew R., Johnson, Eric A., Nye, Dillon B., Lecomte, Juliette T.J.
• Format: Article
• Language: English
• Subjects: Nitric oxide dioxygenase; Nitric oxide reductase; Nitrite reductase; Nitrosyl hydride; Nitroxyl; Truncated hemoglobin
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2017.09.018

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme–histidine C–N linkage similar to the C–S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII–NO) that resist reductive nitrosylation to the paramagnetic FeII–NO forms. Dithionite reduction of FeIII–NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII–NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII–H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine–heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation. The reactivity of hemoglobin toward NO depends on the histidine–heme covalent linkage. In the crosslinked state, the ferrous globin binds NO to form a stable six-coordinate complex; in contrast, NO binding to the unmodified form inhibits crosslinking, causes heme dissociation, and, remarkably, undergoes reduction to produce HNO. [Display omitted] •Synechococcus hemoglobin exists with and without a heme–protein crosslink.•Both forms exhibit nitric oxide dioxygenase activity.•Ferrous crosslinked protein binds NO stably.•Ferrous non-crosslinked protein loses heme on NO binding and generates HNO on reduction.•A protective role for the histidine–heme crosslink is proposed.