Abstract
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) 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 (α and β), while H105 and H134 are conserved only in the β subunits (β1 and β2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC β1(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 β1 subunit is the heme proximal ligand.
| Original language | English |
|---|---|
| Pages (from-to) | 4502-4509 |
| Number of pages | 8 |
| Journal | Biochemistry |
| Volume | 37 |
| Issue number | 13 |
| DOIs | |
| State | Published - 31 Mar 1998 |
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Identification of histidine 105 in the β1 subunit of soluble guanylate cyclase as the heme proximal ligand. / Zhao, Yunde; Schelvis, Johannes; Babcock, Gerald T.; Marletta, Michael A.
In: Biochemistry, Vol. 37, No. 13, 31.03.1998, p. 4502-4509.Research output: Contribution to journal › Article
TY - JOUR
T1 - Identification of histidine 105 in the β1 subunit of soluble guanylate cyclase as the heme proximal ligand
AU - Zhao, Yunde
AU - Schelvis, Johannes
AU - Babcock, Gerald T.
AU - Marletta, Michael A.
PY - 1998/3/31
Y1 - 1998/3/31
N2 - 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) 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 (α and β), while H105 and H134 are conserved only in the β subunits (β1 and β2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC β1(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 β1 subunit is the heme proximal ligand.
AB - 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) 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 (α and β), while H105 and H134 are conserved only in the β subunits (β1 and β2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC β1(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 β1 subunit is the heme proximal ligand.
UR - http://www.scopus.com/inward/record.url?scp=0032584316&partnerID=8YFLogxK
U2 - 10.1021/bi972686m
DO - 10.1021/bi972686m
M3 - Article
C2 - 9521770
AN - SCOPUS:0032584316
VL - 37
SP - 4502
EP - 4509
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 13
ER -