A Structural Study Of Carbon Monoxide Bound Soluble Guanylate Cyclase H-NOX Domains

Soluble Guanylate Cyclase, short for s GC, is an enzyme containing heme group, which function as a signal-massager in mammal NO-c GMP signal pathways. s GC could catalyze GTP into second massager c GMP, regulate metabolic systems and activities such as cardiovascular system, nervous system and cell apoptosis. s GC is composed of a pair of homodimer, which can be divided into two subunits, an α subunit without heme and a β subunit with heme. The smallest fragment in β subunit which could hold a heme group steadily is the first 194 amino acids in N-terminus, short for s GC β1_1-194 H-NOX. Function as an NO receptor, the gas ligate property of s GC is of great significance. All along, the purification of s GC is very difficult for the limitation of enzyme materials, which could only be extracted from animal organs and tissues, also it will take a long time but purified only a little amount. With the development of technology and the discovery of H-NOX family proteins, which are highly conserved and share a highly sequence homology to s GC, the expression and purification of H-NOX protein family and s GC β1 subunits fragments with appropriate host and vector can be achieved. In this H-NOX family, Ns H-NOX is the one with the highest homology to s GC, and together with s GC β1_1-194 H-NOX for their similar gas ligate property to s GC, they become the new focus of model protein in investigation of the relationship between the structure and function of s GC.In this research, we constructed the prokaryotic expression system of s GC β1_1-194 H-NOX(p ET-20b+s GC β1_1-194) and Ns H-NOX（p UC19+anaerobic promoter+hnox） with E. coli as expression host, and we got s GC β1_1-194 H-NOX and Ns H-NOX with higher purity.Since Ns H-NOX and s GC β1_1-194 H-NOX shares a highly gas ligate property with native s GC, we employed resonance Raman spectroscopy（r RS）, Molecular Dynamics Simulations（MD） and Density Functional Theory（DFT） to discuss the binding properties of carbon monoxide（CO） to H-NOX domains and the structural changes of CO bound H-NOX domains.In the resonance Raman Spectroscopy study, we found that both of Ns H-NOX and s GC β1_1-194 H-NOX exhibit a similar two different Fe-CO stretching vibration mode, like the native s GC case, which correspond to two different CO bound mode, that is two different CO bound structures. By comparing the Raman spectra of different truncated s GC β1 subunits, native s GC and Ns H-NOX, with Gaussian fitting peakdifferentiation, we found that the v Fe-CO appears at 494cm-1 and 470cm-1, whereas s GC β1_1-194 H-NOX poison at 489cm-1and 473cm-1 and the relative composition of the two components varies with different truncated s GC β1 subunits. Which further illustrated that every v Fe-CO vibrational mode is correspond to a different CO bound structure, and these two structure components varies in different H-NOX domains.In previous work, we have modeled a 3D structure of s GC β1 H-NOX, using the crystal structure of Ns H-NOX as the template. We discussed the structure changes when CO bound to s GC β1_1-194 H-NOX and Ns H-NOX, according to the 400 ns simulation of MD. In the first 100 ns MD simulation, both proteins experienced large fluctuations before reaching equilibrium after 150 ns. By analyzing our MD trajectories, we found that the equilibrated structures of CO-s GC β1_1-194 H-NOX demonstrated a large shifting motion of the heme group compared with that of Ns H-NOX. This shift is primarily modulated by a conformational bending of the C-terminus αF segment, where the residue H105 is located. Upon further inspection of the structure, we found the bending of αF is caused by a stable formation of a hydrogen bond between Y112 at the end of αF and the conserved Y83 within the hinge region between αD and αE. However, in the case of Ns H-NOX, the residue at position 112 is F, and therefore, no hydrogen bond is formed between the two residues in Ns H-NOX. Moreover, the shifting motion of heme in s GC β1_1-194 H-NOX also leads to a structural change in the protein which opens a portal and allows water access to the distal heme pocket. As a result, the water molecule can form a hydrogen bond with bound CO in the case of s GC β1_1-194 H-NOX. Water molecules can hardly approach the distal heme pocket to form a hydrogen bond with CO in the case of Ns H-NOX, although there are a small number of water molecules which occasionally enter the distal pocket, water would be expelled rapidly due to the short path of the Y-shaped tunnel in the Ns H-NOX. Conversely, in the case of s GC β1_1-194 H-NOX, interactions between CO oxygen and water hydrogen are observed for a large population after 150 ns of our MD simulation. And the distal heme pocket is exposing to the solvent in s GC β1_1-194 H-NOX, and the greater exposure of the distal heme environment of s GC β1_1-194 H-NOX to the solvent also explains the fast oxidation rate of the truncated s GC β1 subunit compared to Ns H-NOX.In order to verify our hypothesize that water entering into the distal heme pocket alters the ligate CO structure, DFT method was employed to calculate the Raman spectra of（Im H） Fe P（CO） system in the presence of water or not. And the calculation results are in agreement with the resonance Raman Spectra we collected. We discussed the influence of different hydrogen bond donor with different polarity on（Im H）Fe P（CO） system, such as H2 S, NH3, H2 O and HF, together with the orbit energy level distribution and electron destiny distribution. In the presence of water hydrogen bond, on the porphyrin macrocycle plane, the electron around N atom decreased, and the electron around Fe atom increased. Whereas on the axis direction, the electron density between C atom and Fe atom increased slightly, which indicates a strong interation between the two atoms, thus reflect on the increased v Fe-CO in Raman spectra. And a feed-back bond formed during this process, the electron transfer from dπ orbit of Fe atom to π* orbit of C atom in CO, along with the increase of hydrogen bond, the electron destiny decrease, and the interaction between C atom and O atom of CO become weaker, resulting in a decrease in v C-O.The reason why there are two different v Fe-CO stretching modes is that a portion of the water molecules in the distal heme pocket could form hydrogen bond with CO to change the conformation of the heme pocket, resulting in an additional Fe-CO stretching band at a distinct frequency, that is a Fe-CO…HOH hydrogen bond vibration mode at a higher frequency, and a Fe-CO non-hydrogen bond vibration mode at a lower frequency.Our study on the CO bound s GC β1 H-NOX and Ns H-NOX is of great significant in clarifying the activation mechanism of s GC and the relationship of structure and function of s GC and H-NOX family proteins, also of great potential value in the design of relative cardiovascular drugs.