Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand.
Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues 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 (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(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 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 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 beta1 subunit is the heme proximal ligand.
Zhao
Y
Y
Department of Biological Chemistry, School of Medicine, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
Schelvis
J P
JP
Babcock
G T
GT
Marletta
M A
MA
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D007093:Imidazoles; D008024:Ligands; D006418:Heme; D006639:Histidine; C029899:imidazole; D006162:Guanylate Cyclase; D000409:Alanine; C018100:1-methylimidazole