Heme stoichiometry of heterodimeric soluble guanylate cyclase.
The soluble form of guanylate cyclase (sGC) is to date the only definitive receptor for the novel signaling agent nitric oxide (.NO). .NO increases the Vmax of sGC by 100-200-fold, and it has been proposed that this activation occurs subsequent to the binding of .NO to a heme moiety on the enzyme. It has previously been demonstrated that the enzyme can be purified in a state containing as much as 1 heme per heterodimer. However, since the two subunits of the heterodimer display considerable homology, and the enzyme routinely loses heme upon purification, it has been unclear whether the native heme stoichiometry is 1 per heterodimer or 2 per heterodimer. Using a novel procedure, the enzyme has been purified to homogeneity from bovine lung in a state containing 1.52 +/- 0.10 equiv of heme per heterodimer, indicating that the native heme stoichiometry is 2 per heterodimer. The .NO-activated specific activity of this enzyme is increased by 50% over that of enzyme containing 1 heme per heterodimer and is the highest specific activity ever observed for sGC. Spectrally only one type of heme is observed, indicating that both hemes in the heterodimer are in similar environments. It is concluded that each subunit of the heterodimer binds 1 equiv of heme at a site conserved between the two subunits. Alignment of the nine published cDNA sequences for sGC indicates that the heme binding domain is the central portion of each subunit, corresponding to residues 213-370 in the bovine beta 1 sequence.
Stone
J R
JR
Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109-1065, USA.
Marletta
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000032:analysis* / Q000378:metabolism; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D013053:Spectrophotometry
D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase