IDENTIFICATION OF HISTIDINE-105 IN THE BETA-1 SUBUNIT OF SOLUBLE GUANYLATE-CYCLASE AS THE HEME PROXIMAL LIGAND

Soluble guanylate cyclase isolated from bovine and rat lung is a heter odimeric hemoprotein composed of alpha 1 and beta 1 subunits. The heme binding region has been localized to residues 1-385 of the beta 1 sub unit [beta 1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine re sidues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta 1 and beta 2). Site-directe d mutagenesis was used to individually change each of the conserved hi stidines in sGC beta 1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105 A and H105G had heme bound as isolated. Imidazole (Im) was able to res cue heme binding to H105G when added to the growth medium and purifica tion buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6- coordinate, low-spin complexes. After reduction, the ferrous heme in H 105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman s pectroscopy. When imidazole in H105G(Im) was exchanged with N-methylim idazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted fro m 221 to 212 cm(-1). A shift of this magnitude is expected when the li gand is directly coordinated to the heme iron. All of the data are con sistent with the conclusion that H105 in the beta 1 subunit is the hem e proximal ligand.