HEME STOICHIOMETRY OF HETERODIMERIC SOLUBLE GUANYLATE-CYCLASE

The soluble form of guanylate cyclase (sGC) is to date the only defini tive receptor for the novel signaling agent nitric oxide ((NO)-N-.). ( NO)-N-. increases the V-max of sGC by 100-200-fold, and it has been pr oposed that this activation occurs subsequent to the binding of (NO)-N -. 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 he me per heterodimer. However, since the two subunits of the heterodimer display considerable homology, and the enzyme routinely loses heme up on purification, it has been unclear whether the native heme stoichiom etry is 1 per heterodimer or 2 per heterodimer. Using a novel procedur e the enzyme has been purified to homogeneity from bovine lung in a st ate containing 1.52 +/- 0.10 equiv of heme per heterodimer, indicating that the native heme stoichiometry is 2 per heterodimer. The (NO)-N-. -activated specific activity of this enzyme is increased by 50% over t hat nf enzyme containing 1 heme per heterodimer and is the highest spe cific activity ever observed for sGC. Spectrally only one type of heme is observed, indicating that both hemes in the heterodimer an in simi lar environments. It is concluded that each subunit of the heterodimer binds 1 equiv of heme at a site conserved between the two subunits. A lignment of the nine published cDNA sequences for sGC indicates that t he heme binding domain is the central portion of each subunit correspo nding to residues 213-370 in the bovine beta(1) sequence.