FUNCTIONS OF CONSERVED CYSTEINES OF SOLUBLE GUANYLYL CYCLASE

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme , is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considere d to be important as they were thought to play a significant role in t he regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen c onserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes we re able to synthesize cGMP. Mutation of two cysteines located in the N -terminal, putative heme-binding region of the beta(1) subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with he me and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta(1) subunit (C ys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of t he corresponding cysteines on the beta(1) subunit did not alter NO res ponsiveness, indicating that heme-binding is mainly a feature of the N -terminal domain of the beta(1) subunit.