A FUNCTIONAL HEME-BINDING SITE OF SOLUBLE GUANYLYL CYCLASE REQUIRES INTACT N-TERMINI OF ALPHA(1) AND BETA(1) SUBUNITS

Soluble guanylyl cyclase, a heterodimeric enzyme, is the most importan t intracellular target for the signalling molecule nitric oxide (NO). NO stimulates the enzyme by binding to a prosthetic heme group. The id entity of the axial heme ligand, however, is still unknown, Here we sh ow that guanylyl cyclase mutated at the residue His105 on the beta(1) subunit, a mutant that we have shown before to contain no heme after p urification [Wedel, B., Humbert, P., Harteneck, C., Foerster, J., Malk ewitz, J., Bohme, E., Schultz, G. & Koesling, D. (1994) Proc Natl Acad Sci. USA 91, 2592-2596] can be reconstituted with heme. The reconstit uted mutant remains NO-insensitive and displays an ultraviolet absorpt ion spectrum consistent with an altered axial coordination. Thus, this residue is a strong candidate for the axial heme-ligating residue and appears to be necessary for NO stimulation. Apart from the axial heme ligand, the role of the enzyme's two subunits, alpha(1) and beta(1), in heme binding has not been clarified to date. To address this questi on, we purified mutant heterodimers in which the non-conserved amino t ermini of either alpha(1) (131 residues deleted): or beta(1) (64 resid ues) have been deleted. These deletion mutants had previously been fou nd to be marginally (alpha(1) truncated) or not at all NO sensitive (b eta(1) truncated) in cytosolic fractions [Wedel, B., Harteneck, C., Fo erster, J., Friebe, A., Schultz, G. & Koesling, D. (1995) J. Biol. Che m. 270, 24871-24875]. Here, we show that the purified enzyme truncated on alpha(1) has a significantly reduced capacity to bind heme which e xplains the reduced NO sensitivity. By contrast, the beta(1)-truncated enzyme binds an amount of heme comparable to the wird type but is onl y marginally NO-responsive and displays a shift in the heme ultraviole t absorption maximum indicative of altered heme coordination. In concl usion, the heme binding site of soluble guanylyl cyclase requires the presence of both subunits in full length to be able to bind wild-type quantities of heme and to be capable of mediating the NO-heme-induced stimulation. Despite some structural similarity, both subunits appear to participate differently in NO-heme-mediated enzyme regulation.