Volume 5 Supplement 1

2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications

Open Access

Resonance raman investigation of effects of YC-1 and GTP on structure of CO-bound heme of soluble guanylate cyclase

  • Biswajit Pal1,
  • Zhengqiang Li1, 3, 4,
  • Shigeo Takenaka2,
  • Shingo Tsuyama2 and
  • Teizo Kitagawa1Email author
BMC Pharmacology20055(Suppl 1):S3

DOI: 10.1186/1471-2210-5-S1-S3

Published: 16 June 2005

Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been categorized into two types; sGC1 and sGC2. Here we demonstrate that the RR spectra of sGC isolated from bovine lung contains only sGC2 while both species are observed in the spectra of CO-bound form (CO-sGC). The relative populations of the two forms altered from an initial composition in which the CO-sGC2 form predominated, with the Fe-CO (νFe-CO) and C-O stretching modes (νCO) at 472 and 1985 cm-1, respectively, to a composition dominated by the CO-sGC1 form with νFe-CO and νCO at 488 and 1969 cm-1, following the addition of xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a similar but significantly smaller effect, while a substrate analogue, GTP-γ-S had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced.

In the absence of any effectors, the Fe-CO stretching and the heme vinyl bending modes appeared at 473 and 424 cm-1, respectively. There was no band around 372 cm-1. However, in the presence of YC-1 and GTP, the Fe-CO stretching mode was shifted to 489 cm-1 and the vinyl mode appeared at 400 cm-1. The propionate mode appeared at 372 cm-1 as a sharp band. The vinyl modes at 424 and 400 cm-1 are assigned to the vinyl-2 and vinyl-4 Cβ - Ca = Cb bending vibrations that are strongly coupled with ring modes of pyrroles-I and II, respectively, while the propionate mode is coupled with ring modes of pyrroles-III and IV. In addition, new CO-isotope sensitive bands were observed at 521, 363, and 227 cm-1. The 521 cm-1 band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron-protoporphyrin IX in CTAB micelles. Distinct from the 472 cm-1 species, both the 488-cm-1 and 521-cm-1 species were apparently unphotodissociated when an ordinary Raman spinning cell was used with a CW laser source for Raman excitation, indicating rapid recombination of photodissociated CO in the presence of YC-1 and GTP. On the basis of these findings, binding of YC-1 and GTP to the heme pocket is proposed.

Authors’ Affiliations

(1)
Okazaki Institute for Integrative Bioscience, National Institute of Natural Sciences
(2)
Department of Veterinary Science, Osaka Prefecture University
(3)
Centre for Cellular and Molecular Biology
(4)
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University

Copyright

© BioMed Central Ltd 2005

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