Interaction of soluble guanylate cyclase with YC-1: Kinetic and resonance Raman studies

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic ben zylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activate s sGC approximately 5-fold, YC-1 activates sCC to a level comparable to sti mulation by NO alone. We have used kinetic analyses and resonance Raman spe ctroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 mu M YC-1, the V-max/K-m (GTP) incr eases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the GO-bound form from 474 to 492 cm(-1). Similar ly, YC-1 has no effect on the RR spectrum of ferrous beta 1(1-385), the iso lated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta 1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the GO-bound forms of sGC and beta 1(1-385) in the pre sence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proxima l ligands with imidazole character, which suggests that histidine remains t he heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does n ot shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescue d heme ligand mutant of beta 1(1-385). The data are consistent with binding of CO and YC-1 to the sGC hemebinding domain leading to conformational cha nges that give rise to an increase in catalytic turnover and a change in th e electrostatic environment of the heme pocket.