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

Abstract

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.

Towards the understanding of the enzymatic cleavage of polyisoprene by the dihaem-dioxygenase RoxA

“…As expected, exposure of RoxA-Wt to nitrogen monoxide gas changed the optical spectrum resulting in new signals at 420, 530 and 562 nm, which resemble spectra of NO adducts of other haem proteins (e.g. Moir 1999; Herold and Rehmann 2003; Turner et al 2005; Preimesberger et al 2017). In analogy, we expected similar but distinguishable signals for Fe 2+ –NO and Fe 3+ –NO adducts.…”

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

“…Two reduction systems were compared. The first was composed of the heme-free diaphorase domain of C. reinhardtii NIT1, which was prepared recombinantly as described previously [10]. The second was a crude cell extract of partially purified NIT1.…”

“…For multiple NO addition experiments, MAHMA-NONOate was introduced after nearly full recovery to the Fe(II)–O 2 form. This protocol differs from that originally used for THB1 (Fd-FNR system with glucose-6-phosphate (G6P) and G6P dehydrogenase to regenerate NADPH from NADP + [3]) and is identical to that used in a Synechococcus GlbN study [10] except for a higher concentration of FNR.…”

“…If Lys E10 assists in reactions that demand both redox chemistry and substrate binding, His–Fe–Lys coordination is not the only scheme up to the task. This is illustrated by variants of THB1 lacking this residue [3] and relatives of THB1 having His–Fe ligation or His–Fe–His ligation, which are all capable of some NOD activity [9, 10]. Furthermore, a histidine at position E10 can also serve as a ligand in both iron oxidation states, presumably at a lower energetic cost than a lysine because the p K a of histidine is close to physiological pH.…”

Reannotation of the Ribonucleotide Reductase in a Cyanophage Reveals Life History Strategies Within the Virioplankton

“…The tyrosine residue harbors a stable protein radical and is a target of nitric oxide (Eiserich et al, 1995; Radi, 2004). Tyrosine-radical scavenging nitric oxide is hypothesized to be present inside Synechococcus cells as an intermediate in nitrate reduction (Preimesberger et al, 2017), which is widespread among freshwater and marine Synechococcus species and is coupled to photosynthesis (Guerrero, 1985; González et al, 2006; Klotz et al, 2015; Sunda and Huntsman, 2015). Thus, the loss of the tyrosine radical site in the Class I β subunit genes of cyanophage, such as P-SSP7, would enable these phages to avoid RNR inactivation by nitric oxide.…”