Incorporation of tyrosine and glutamine residues into the soluble guanylate cyclase heme distal pocket alters NO and O2 binding.
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.
Department of Molecular and Cell Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Emily R
ER
Derbyshire
Sarah
S
Deng
Michael A
MA
Marletta
D000595:Amino Acid Sequence; D000818:Animals; D005973:Glutamine / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006162:Guanylate Cyclase / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D000071756:Soluble Guanylyl Cyclase; D013053:Spectrophotometry / Q000379:methods; D014443:Tyrosine / Q000737:chemistry*
D011166:Porphyrins; D018160:Receptors, Cytoplasmic and Nuclear; D005973:Glutamine; D009569:Nitric Oxide; D014443:Tyrosine; D006418:Heme; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D010100:Oxygen