Identification of Histidine 105 in the 1 Subunit of Soluble Guanylate Cyclase as the Heme Proximal Ligand
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- 作者：Yunde Zhao ; Johannes P. M. Schelvis ; Gerald T. Babcock ; and Michael A. Marletta
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：March 31, 1998
- 年：1998
- 卷：37
- 期：13
- 页码：4502 - 4509
- 全文大小：86K
- 年卷期：v.37,no.13(March 31, 1998)
- ISSN：1520-4995
文摘
Soluble guanylate cyclase isolated from bovine and rat lung is aheterodimeric hemoproteincomposed of 
1 and 
1 subunits. The heme binding region hasbeen localized to residues 1-385 of the1 subunit [1(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 bindingregion of sGC. H220 and H346 areconserved among all known sGC subunits ( and ), while H105 andH134 are conserved only in the subunits (1 and 2). Site-directed mutagenesis was used toindividually change each of the conservedhistidines in sGC 1(1-385) to alanine or glycine, and theresulting mutants were expressed in E. coli.All of the mutants except for H105A and H105G had heme bound asisolated. Imidazole (Im) was ableto rescue heme binding to H105G when added to the growth medium andpurification buffers. The hemein H105G isolated in the presence of imidazole [H105G(Im)] wasferric and a mixture of 5-coordinate,high-spin and 6-coordinate, low-spin complexes. After reduction,the ferrous heme in H105G(Im) was5-coordinate, high-spin as indicated by resonance Raman spectroscopy.When imidazole in H105G(Im)was exchanged with N-methylimidazole (MeIm), theFe-N(Im/MeIm) stretching frequency was shiftedfrom 221 to 212 cm-1. A shift of thismagnitude is expected when the ligand is directly coordinatedtothe heme iron. All of the data are consistent with the conclusionthat H105 in the 1 subunit is the hemeproximal ligand.
1 and 1 subunits. The heme binding region hasbeen localized to residues 1-385 of the1 subunit [1(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 bindingregion of sGC. H220 and H346 areconserved among all known sGC subunits ( and ), while H105 andH134 are conserved only in the subunits (1 and 2). Site-directed mutagenesis was used toindividually change each of the conservedhistidines in sGC 1(1-385) to alanine or glycine, and theresulting mutants were expressed in E. coli.All of the mutants except for H105A and H105G had heme bound asisolated. Imidazole (Im) was ableto rescue heme binding to H105G when added to the growth medium andpurification buffers. The hemein H105G isolated in the presence of imidazole [H105G(Im)] wasferric and a mixture of 5-coordinate,high-spin and 6-coordinate, low-spin complexes. After reduction,the ferrous heme in H105G(Im) was5-coordinate, high-spin as indicated by resonance Raman spectroscopy.When imidazole in H105G(Im)was exchanged with N-methylimidazole (MeIm), theFe-N(Im/MeIm) stretching frequency was shiftedfrom 221 to 212 cm-1. A shift of thismagnitude is expected when the ligand is directly coordinatedtothe heme iron. All of the data are consistent with the conclusionthat H105 in the 1 subunit is the hemeproximal ligand.