Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Abstract

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC).The heme cofactor of ␣1␤1 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 ␤1(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 fulllength sGC we mutated several conserved distal heme pocket residues (␤1 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 ␤1 distal heme pocket (Ile-145 3 Tyr and Ile-149 3 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 ␣1␤1 protein. Additionally, these data suggest that atypical sGCs stabilize O 2 binding by a hydrogen bonding network involving tyrosine and glutamine.Soluble guanylate cyclase (sGC) 3 is the most thoroughly characterized receptor for the gaseous signaling agent nitric oxide (NO). NO induced activation of sGC is critical to several physiological processes, including neurotransmission, vasodilation, and platelet aggregation (1-3). The importance of sGC to physiological function has been clearly demonstrated over the last decade (4, 5); however, much less is understood about the molecular mechanisms that regulate enzyme activity.sGC is a heterodimeric hemoprotein consisting of two homologous subunits, ␣ and ␤. The ␣1␤1 heterodimer is the most prevalent and commonly studied protein. Despite the same histidine ligated heme and iron oxidation state as found in the globins, sGC shows no measurable affinity for O 2 and therefore can selectively bind NO in the presence of O 2 (reviewed in Refs. 6, 7). Interestingly, not only does sGC discriminate against O 2 binding to the heme, but both the Fe II -unligated and Fe II -NO species are stable in an aerobic environment (8). This is in stark contrast to other hemoproteins that readily bind and ultimately react with O 2 (9 -12). The mechanism by which sGC discriminates against O 2 binding as well as the molecular events that lead to activation remain to be elucidated.There have been several proposals on the mechanism of ligand discr...

Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase

“…Also, the kinetics of NO dissociation from rat heme domain ␤ 1 (27) and kinetics of NO binding to bacterial Nostoc NO sensor (sequence 1-183) (13) are different from those of entire sGC. This conformational difference may also explain the observation that the same single mutation (I145Y) does not induce O 2 binding affinity in the full-length sGC (10,28) as it does in the ␤ 1 (1-385) construct (29), whereas a homologous bacterial NO sensor possesses a high O 2 affinity (30).…”

Probing Domain Interactions in Soluble Guanylate Cyclase

“…The change in the absorbance maximum versus time was plotted and the data were fit to a single exponential equation. Dissociation of NO from the heme of sGC was measured at 25 °C using the CO/dithionite trapping method described previously (17). The change in the absorbance maximum versus time was plotted and the data were fit to a double exponential equation.…”

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain

“…The presence of a distal pocket tyrosine residue has been shown to be important for stabilizing O 2 binding in the H-NOX family (25). However, it is apparent that additional structural and electronic features contribute to ligand-binding properties (25)(26)(27)(28)(29)(30).…”