Localization of the Heme Binding Region in Soluble Guanylate Cyclase
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- 作者：Yunde Zhao and Michael A. Marletta
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 16, 1997
- 年：1997
- 卷：36
- 期：50
- 页码：15959 - 15964
- 全文大小：231K
- 年卷期：v.36,no.50(December 16, 1997)
- ISSN：1520-4995
文摘
Soluble guanylate cyclase (sGC) is a heterodimeric hemoproteincomposed of 
1 and 
1subunits. sGC is activated by nitric oxide (NO) and thereforeplays a central role in NO signal transduction.Activation of sGC by NO is believed to be mediated by theinteraction between NO and the heme ofsGC. Spectroscopic and kinetic studies have shown that the heme ofsGC is in a unique environment.Characterization of the heme environment is critical to theunderstanding of the mechanism of NO activation.To approach this goal, the 1 N-terminal fragment consisting ofresidues 1-385 [1(1-385)] of sGCwas expressed in E. coli. 1(1-385) was thenpurified to homogeneity in two steps by DEAE ion exchangeand gel filtration chromatography. Purified 1(1-385) wasfound to contain a stoichiometric amount ofheme. The UV-visible spectrum of 1(1-385) is almostidentical to that of the native heterodimericsGC purified from bovine lung. 1(1-385) binds both NOand CO, leading to a shift in the Soret maximumfrom 431 nm to 398 and 423 nm, respectively. These spectral shiftsare identical to those observed withheterodimeric sGC purified from bovine lung. These results suggestthat the heme in the 1(1-385) issimilar to that in the heterodimeric sGC. Therefore, for the firsttime, the heme binding region of sGChas been unambiguously localized to the N-terminal region of the 1subunit. Our data also suggest thatthe N-terminal region of the 1 subunit of sGC is itself sufficientfor heme binding.
1 and 1subunits. sGC is activated by nitric oxide (NO) and thereforeplays a central role in NO signal transduction.Activation of sGC by NO is believed to be mediated by theinteraction between NO and the heme ofsGC. Spectroscopic and kinetic studies have shown that the heme ofsGC is in a unique environment.Characterization of the heme environment is critical to theunderstanding of the mechanism of NO activation.To approach this goal, the 1 N-terminal fragment consisting ofresidues 1-385 [1(1-385)] of sGCwas expressed in E. coli. 1(1-385) was thenpurified to homogeneity in two steps by DEAE ion exchangeand gel filtration chromatography. Purified 1(1-385) wasfound to contain a stoichiometric amount ofheme. The UV-visible spectrum of 1(1-385) is almostidentical to that of the native heterodimericsGC purified from bovine lung. 1(1-385) binds both NOand CO, leading to a shift in the Soret maximumfrom 431 nm to 398 and 423 nm, respectively. These spectral shiftsare identical to those observed withheterodimeric sGC purified from bovine lung. These results suggestthat the heme in the 1(1-385) issimilar to that in the heterodimeric sGC. Therefore, for the firsttime, the heme binding region of sGChas been unambiguously localized to the N-terminal region of the 1subunit. Our data also suggest thatthe N-terminal region of the 1 subunit of sGC is itself sufficientfor heme binding.