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 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.

FIGURE 1.

Alignment of NO-activated sGCs with predicted O₂-binding sGCs. Numbering is that of the rat β1 protein. Structure of Tt H-NOX (left) and homology model of O₂-binding Gyc-88E H-NOX domain (right) are shown. The Tt H-NOX structure shows that O₂ is stabilized at the heme by a hydrogen bonding network involving Trp-9, Asn-74, and Tyr-145 (1U55.pdb) (W9, N74, and Y140 in the Tt H-NOX numbering system). The homology model of the 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).

FIGURE 2.

Electronic absorption spectra of sGC β1 distal pocket mutants at 20 °C. sGC Fe^II-unligated (black solid line), Fe^II-CO (black dashed line), and Fe^II-NO (gray solid line) complexes are shown for wild-type α1β1, α1β1 V5Y, α1β1 F74Y, α1β1 I145Y, α1β1 I149Y, and α1β1 I145Y/I149Q. All single point mutants do not bind O₂ or oxidize after exposure to air.

FIGURE 3.

Effect of GTP and YC-1 on the temperature-dependent equilibrium between 5- and 6-coordinate Fe^II-NO complexes in sGC α1β1 I145Y and α1β1 F74Y. As the temperature increases from 5 to 50 °C the population of 6-coordinate sGC Fe^II-NO (416 nm) decreases and the population on 5-coordinate sGC Fe^II-NO increases (399 nm). Spectra are shown for 5 (black solid line), 30 (black dashed line), and 50 °C (gray solid line) for α1β1 I145Y and for 5 (black solid line), 20 (black dashed line), and 30 °C (gray solid line) for α1β1 F74Y. GTP and YC-1 lead to a greater population of the 5-coordinate Fe^II-NO complex.

SCHEME 1

FIGURE 4.

Time courses for observed NO dissociation from sGC β1 distal pocket mutants in the presence of a CO/dithionite trap at 25 °C. Data were extracted from difference spectra and plotted with a double (solid line) exponential fit. Wild-type α1β1, α1β1 V5Y, α1β1 F74Y, α1β1 I145Y, and α1β1 I149Y were at 1 μm. The time courses shown are the average of duplicate dissociation experiments repeated two to four times for each construct. The low ΔAbs for the α1β1 I149Y mutant is due to a very slow NO dissociation rate.

FIGURE 5.

Activity of sGC β1 distal pocket mutants in the absence and presence of CO and YC-1 at 37 °C. The bars, from left to right, refer to wild-type α1β1, α1β1 V5Y, α1β1 F74Y, α1β1 I145Y, and α1β1 I149Y. All constructs form 6-coordinate complexes with CO, but the Fe^II-CO complexes are activated to varying degrees by the presence of YC-1 (150 μm).

FIGURE 6.

Reaction of sGC α1β1 I145Y/I149Q with O₂. Left panel, reaction of sGC (500 nm) with O₂ at 10 °C. Once O₂ is added to the protein the absorbance maximum immediately shifts from 426 nm (black trace) to 417 nm (dark gray trace). Over time the 417 nm species converts to a 413 nm species (light gray trace). The time between each trace was 12 s. Right panel, the change in absorbance versus time was plotted, and data were fit to a single exponential equation.