Resonance Raman and infrared spectroscopic studies of high-output forms of human soluble guanylyl cyclase.
The allosteric regulator BAY-41-2272 converts the CO adduct of soluble guanylyl cyclase (CO-sGC) enzyme from a low- to high-output form, with respect to production of cGMP. Resonance Raman (RR) and Fourier Transform Infrared (FTIR) spectroscopic techniques are used to show that the CO-sGC exists as major and minor conformers, both having nu(Fe-CO) and nu(C-O) modes characteristic of 6-coordinate species. It is further shown that addition of BAY-41-2272 to the CO adduct induces the transition of some fraction of the initial CO-heme adducts into two new CO-heme complexes, the fractional conversion being dependent on the temperature. One new complex displays vibrational modes characteristic of pentacoordinated CO-adduct, and its formation is not affected by temperature. The second complex, although slightly different from the original CO-adducts, is hexacoordinated, and its formation is facilitated by temperature. The production of substantial amounts of the 5-coordinate CO adduct upon addition of BAY-41-2272, reveals the fact that several out-of-plane heme deformation modes are simultaneously activated, an observation similar to that realized upon NO activation. While the precise nature of these modes will require elucidation by isotopic labeling experiments, by analogy with earlier studies of other heme proteins, several bands associated with modes attributable to peripheral substituent deformations and methine carbon movements are implicated. The documented formation of two new forms upon addition of Bay-41-2272 (a 5-coordinate and a new 6-coordinate form) is discussed with respect to the implications for enzyme activation.
Martin
Emil
E
Department of Integrative Biology and Institute of Molecular Medicine, University of Texas Houston Medical School, Houston, TX 77030, USA.
Czarnecki
Kazimierz
K
Jayaraman
Vasanthi
V
Murad
Ferid
F
Kincaid
James
J
D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007191:Indazoles / Q000737:chemistry / Q000494:pharmacology; D007527:Isoenzymes; D011487:Protein Conformation; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship
C423582:3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine; D007191:Indazoles; D007527:Isoenzymes; D011720:Pyrazoles; D011725:Pyridines; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D006418:Heme; D002248:Carbon Monoxide; D006162:Guanylate Cyclase