Abstract
Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha1beta1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta1 distal heme pocket (Ile-145 --> Tyr and Ile-149 --> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe(II)-O(2) complexes and is likely the first observation of a Fe(II)-O(2) complex in the full-length alpha1beta1 protein. Additionally, these data suggest that atypical sGCs stabilize O(2) binding by a hydrogen bonding network involving tyrosine and glutamine.
Highlights
Soluble guanylate cyclase3 is the most thoroughly characterized receptor for the gaseous signaling agent nitric
This proposal was called into question in recent reports that showed that the introduction of a tyrosine in the 1 subunit at a position that aligns with Thermoanaerobacter tengcongensis (Tt) hemenitric oxide and oxygen (H-NOX) Tyr145 in full-length Soluble guanylate cyclase (sGC) does not yield an O2-binding protein [17, 18] despite the fact that this point mutant in the sGC hemebinding construct 1(1–385) was able to bind O2 [13]
These atypical O2-binding sGCs, like Gyc-88E, lack the tryptophan and asparagine known to be important for O2 binding in Tt H-NOX, multiple sequence alignments suggest another possible hydrogen bond donor is present in the heme distal pocket: glutamine
Summary
O2-binding Gyc-88E suggests that residues capable of stabilizing O2 binding, including Tyr-145 and Gln-149, are in the distal heme pocket (Y143 and Q147 in the Gyc-88E numbering system). Like some GAF domain-containing proteins (DevS [21] and DosT [22]), and known globin-coupled sensors [23,24,25], this O2-binding guanylate cyclase likely requires this tyrosine for O2 ligation These atypical O2-binding sGCs, like Gyc-88E, lack the tryptophan and asparagine known to be important for O2 binding in Tt H-NOX, multiple sequence alignments suggest another possible hydrogen bond donor is present in the heme distal pocket: glutamine. A homology model of Gyc-88E suggests that this glutamine is in proximity to both the distal pocket tyrosine and O2 bound to the heme This residue is conserved in sGCs that contain a tyrosine in the predicted heme distal pocket. This report evaluates sGC activation after mutagenesis of conserved heme pocket residues that are proposed to play an important role in maintaining the proteins heme conformation [16]
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