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

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. G1bN 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 GIbN 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 (Fe-III-NO) that resist reductive nitrosylation to the paramagnetic Fe-III-NO forms. Dithionite reduction of Fe-III-NO G1bN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected Fen NO complex, NO binding to Fe-II G1bN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining Fe-III G1bN, N-15-labeled nitrite, and excess di-thionite resulted in the formation of Fe-II-(HNO)-N-15 G1bN. 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 GIbN. Overall, the results suggest a role for the covalent modification in Fe-II GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.