The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe+2 ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c4), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c4 (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (Oepsilon1) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and Ob atoms of the Asp 52 of Cyt c4 and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the Nepsilon atom of Lys 81 of RCy. The Oepsilon1 of Glu 61 of Cyt c4 is also H-bonded to Ogamma atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances approximately 2.10-2.95 A) between the Lys 81 (RCy), Glu 61 (Cyt c4), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c4 through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc4) to Glu 126 (CcO) with distances approximately 2.95-3.0 A.
The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study,it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.
Rusticyanin (RCy) mediated transfer of electron to Cytochrome C(4) (Cytc(4)) from the extracellular Fe(+2) ion is primarily involved in the Thiobacillus ferrooxidans induced bio-leaching of pyrite ore and also in the metabolism of this acidophilic bacteria. The modeling studies have revealed the two possible mode of RCy-Cytc(4) complexation involving nearly the same stabilization energy approximately -15 x 10(3) kJ/mol, one through N-terminal Asp 15 and another -C terminal Glu 121 of Cytc(4) with the Cu-bonded His 143 of RCy. The Asp 15:His 143 associated complex (DH) of Cytc(4)-RCy was stabilized by the intermolecular H-bonds of the carboxyl oxygen atoms O(delta1) and O(delta2) of Asp 15 with the Nepsilon-atom of His 143 and O(b) atoms of Ala 8 and Asp 5 (of Cytc(4)) with the Thr 146 and Phe 51 (of RCy). But the other Glu 121:His 143 associated complex (EH) of Cytc(4)-RCy was stabilized by the H-bonding interaction of the oxygen atoms O(epsilon1) and O(epsilon2) of Glu 121 with the Nepsilon and Ogamma atoms of His 143 and Thr 146 of RCy. The six water molecules were present in the binding region of the two proteins in the energy minimized autosolvated DH and EH-complexes. The MD studies also revealed the presence of six interacting water molecules at the binding region between the two proteins in both the complexes. Several residues Gly 82 and 84, His 143 (RCy) were participated through the water mediated (W 389, W 430, W 413, W 431, W 373, and W 478) interaction with the Asp 15, Ile 82, and 62, Tyr 63 (Cytc(4)) in DH complex, whereas in EH complex the Phe 51, Asn 80, Tyr 146 (RCy) residues were observed to interact with Asn 108, Met 120, Glu 121 (of Cytc(4)) through the water molecules W 507, W 445, W 401, W 446, and W 440. The direct water mediated (W 478) interaction of His 143 (RCy) to Asp 15 (of Cytc(4)) was observed only in the DH complex but not in EH. These direct and water mediated H-bonding between the two respective proteins and the binding free energy with higher interacting buried surface area of the DH complex compare to other EH complex have indicated an alternative possibility of the electron transfer route through the interaction of His 143 of RCy and the N-terminal Asp 15 of Cytc(4).
Cysteine protease is ubiquitous in nature. Excess activity of this enzyme causes intercellular proteolysis, muscle tissue degradation, etc. The role of water-mediated interactions in the stabilization of catalytically significant Asp158 and His159 was investigated by performing molecular dynamics simulation studies of 16 three-dimensional structures of plant thiol proteases. In the simulated structures, the hydrophilic W(1), W(2) and WD(1) centers form hydrogen bonds with the OD1 atom of Asp158 and the ND1 atom of His159. In the solvated structures, another water molecule, W(E), forms a hydrogen bond with the NE2 atom of His159. In the absence of the water molecule W(E), Trp177 (NE1) and Gln19 (NE2) directly interact with the NE2 atom of His159. All these hydrophilic centers (the locations of W(1), W(2), WD(1), and W(E)) are conserved, and they play a critical role in the stabilization of His-Asp complexes. In the water dynamics of solvated structures, the water molecules W(1) and W(2) form a water...water hydrogen-bonded network with a few other water molecules. A few dynamical conformations or transition states involving direct (His159 ND1...Asp158 OD1) and water-mediated (His159 ND1...W(2)...Asp158 OD1) hydrogen-bonded complexes are envisaged from these studies.
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