Characterization of the nitric oxide sensing domain of soluble guanylate cyclase.

Nitric oxide (NO), a reactive and toxic free radical, is involved in many physiological processes. The only known physiological NO receptor is soluble guanylate cyclase (sGC), which catalyzes the conversion of GTP to cGMP. In the presence of NO, the reaction catalyzed by sGC is accelerated a few hundred-fold. sGC is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region was shown to be at the N-terminal region of the beta1 subunit [residues 1--385, beta1(1--385)]. Expressed in and purified from E. coli, beta1(1--385)] is a homodimer and contains one heme per monomer. It has identical spectroscopic properties to those of heterodimeric sGC. Histidine 105 in the beta1 subunit was identified as the heme proximal ligand form studies using site-directed mutagenesis, ligand exchange, and resonance Raman spectroscopic approaches. Activation of sGC by NO is mediated by NO binding to the heme moiety in sGC. The unique heme environment of sGC was shown to enable it to be a fast and efficient NO trap with an on-rate of at least 1.4 x 10 8 M-1 s-1 at 4°C. This is at least 1 order of magnitude faster than NO binding to other hemoproteins. This fast on-rate indicates that sGC is able to efficiently trap NO in the presence of other hemoproteins. It was shown that when NO binds to sGC heme, it initially forms a 6-coordinate intermediate that is then converted to a 5-coordinate NO complex by cleavage of the Fe-His bond. This cleavage is necessary and may be the trigger for sGC activation by NO since the 6-coordinate sGC-NO complex is not activated. The rate of the conversion of the inactive 6-coordinate NO complex to the 5-coordinate active NO complex depends on NO concentration. Thus, at any given time, NO concentration not only determines how many molecules of sGC is activated, but also how much time needed for sGC activation. We also found that cleavage of the Fe-His bond is not the only structural changes in the heme pocket. After the cleavage of the Fe-His bond, the proximal histidine moves from its original position so that exogenous imidazole can form a 6-coordinate sGC-NO complex by directly coordinating to the heme Fe. Since the heme binding region is at the N-terminal region and the catalytic domain is at the C-terminal region, the signal of NO binding to sGC heme must be transmitted to the catalytic domain to activate sGC. A highly conserved domain between the heme domain and the catalytic domain may be responsible for such signal transmission. A leucine zipper motif in this domain is important for the interaction of the alpha1 and beta1 subunits and formation of the homodimeric beta1(1--385). Structural changes in the heme pocket induced by NO binding can be transmitted to the central domain to cause to monomerization of beta1(1--385). These findings should help future studies on detailed mechanisms of sGC activation and deactivation.PhDBiochemistryBiological SciencesBiophysicsMolecular biologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132045/2/9938578.pd