Several compounds have been described in the literature

Several compounds have been described in the literature as inhibitors of the sGC. Among them, methylene blue and LY83583 () are not direct sGC inhibitors, but rather block cGMP formation by generating superoxide anion radicals that deactivate NO. Despite their extensive use in plethora of research studies, the off-target effects of these compounds, , limit their pharmacological value in the in vivo evaluation of the sGC activity. More specific inhibition of sGC has been obtained by other inhibitors like 1-[1,2,4]oxadiazolo[4,3-]quinoxaline-1-one (ODQ) and its 8-bromo analogue NS2028. ODQ acts as an oxidant of the heme group, as it has been revealed from Raman spectroscopy, resulting in the desensitization of the sGC towards the activating action of NO. Although ODQ does not affect the activity of related cyclases (for example the particulate guanylate cyclase), its binding affinity to the heme moiety may contribute to the unspecific interaction with other hemoproteins in vivo, leading to decreased enzyme selectivity and undesirable side effects.
Introduction Soluble guanylate cyclase (sGC), the biological Cordycepin for the physiological nitric oxide (NO), is one crucial heme b containing metalloenzyme involved in NO signaling. Upon NO binding to the heme moiety, sGC increases the activity of catalyzing the conversion of substrate GTP to cGMP, further initiates the subsequent amplification of NO-dependent signaling cascades and plays a key role in many physiological processes [1], [2], [3]. sGC in eukaryotes is comprised an α and β subunits with the α1β1 heterodimer as the abundant form in vivo. Heterodimerization is the prerequisite for the sGC activity. However, recently, the α1/α1 and β1/β1 homodimers have been identified to exist in vivo and possess different physiological roles, respectively [4], [5], [6]. Therefore, it is of great significance to study the sGC dimerization, which has never been addressed before. It has been proposed that both the α and the β subunits of sGC consist of an N-terminal heme domain (also termed H-NOX domain), a following Per/Arnt/Sim (PAS)-like domain, coiled-coil helix, and a C-terminal catalytic domain based on the structural domain prediction, although three dimensional structure for the full sGC is not available. Therein, heme domain, responsible for the heme location and NO sensing, is the key region of sGC bioactivity. In our previous work, the heme domain of human sGC β1 subunit (hsGCβ195) was suggested to form stable homodimer [7]. Cysteine residues, particularly important for the structure and function of proteins, might be involved in oxidation–reduction reactions and several posttranslational modifications (nitrosylation, alkylation, acylation and so on) [8], [9], [10]. The conserved cysteines in sGC also received broad attention and were indicated to be involved in sGC regulation of catalytic activity [11], [12], [13], [14]. To our best knowledge so far, the definite influences of cysteines on the sGC dimerization are striking issues but not clearly understood yet.
Materials and methods The heme domain of human sGC β1 subunit (hsGCβ195) was obtained as described previously [7], [15]. The QuickChange Site-Directed Mutagenesis Kit was purchased from QIAGEN. Oligonucleotide PCR primer pairs were synthesized and DNA sequencing was performed by Shanghai Invitrogen Biotech. The Superdex™ 200 16/60 gel filtration column was from Pharmacia. The other reagents were of analytical grade. Three cysteines mutants including C78A, C122A and C174S were prepared with the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The primer pairs (P1, P2 and P3 for C78A, C122A and C174S, respectively) were as follows. The mutated plasmids were verified by gene sequencing. The expression, purification and heme reconstitution of the mutants were conducted as described before [7]. Analytical gel filtration chromatography was carried out on an AKTA FPLC system (GE Healthcare) to study the aggregation state of hsGCβ195 and the cysteine mutations. Proteins were loaded onto a Superdex™ 200 16/60 pre-equilibrated with column buffer (20mmol/L Hepes, pH 7.4, 150mmol/L NaCl).