Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of α1 and β1 subunits. The heme binding region has been localized to residues 1-385 of the β1 subunit [β1(1-385)], while the catalytic site(s) has been localized to the C-terminal region. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (α and β), while H105 and H134 are conserved only in the βsubunits (β1 and β2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC βl (1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole[H105G(Im)] was ferric and a mixture of five-coordinate, high-spin and six-coordinate, low-spin complexes. Upon reduction, the ferrous heme in HlOSG(Im) was five-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with W-methylimidazole (Melm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 cm-1 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the βl subunit is the heme proximal ligand.
|State||Published - 1 Dec 1998|