Effects of GTP on bound nitric oxide of soluble guanylate cyclase probed by resonance Raman spectroscopy.
Soluble guanylate cyclase (sGC) was isolated from bovine lung, and its resonance Raman (RR) spectra were investigated for the reduced, CO-bound (CO-sGC), NO-bound (NO-sGC), oxidized, and oxidized NO-bound forms in the presence and absence of GTP. The enzyme was purified by more than 12 000-fold in terms of specific activity than the supernatant of homogenates, and the heme content was determined with the pyridine hemochoromogen method and Bradford's protein assay to be 0.8 per heterodimer (alpha, Mr = 74 000; beta, Mr = 69 000). The RR spectra of sGC and CO-sGC including the Fe-His stretch at 203 cm-1 and the Fe-CO stretch at 473 cm-1 were unaltered by binding of GTP and cGMP, but apparent RR spectra of NO-sGC in the presence of GTP changed with time and concentrations of GTP. In the absence of GTP, the RR bands of the N-O stretch (nuNO) and the Fe-NO stretch (nuFe-NO) were observed at 1681 and 521 cm-1, respectively. In its presence, however, two nuNO bands were observed at 1700 and 1681 cm-1, which exhibited 15NO isotopic frequency shifts of 32 and 34 cm-1, respectively. Similar Raman spectral changes were observed with the same amount of cGMP but not with PPi or GTP analogues including ATP, GMPPNP, and GTPgammaS. This suggests that GTP or cGMP binds to the distal side of the heme in the proximity of bound NO, possibly regulating NO binding.
Tomita
T
T
The Graduate University for Advanced Studies and Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki, 444 Japan.
Ogura
T
T
Tsuyama
S
S
Imai
Y
Y
Kitagawa
T
T
D000818:Animals; D002417:Cattle; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D008168:Lung / Q000201:enzymology; D015335:Molecular Probes; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods
D015335:Molecular Probes; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase