Rusticyanin (RCy) mediated transfer of electron to Cytochrome C4 (Cytc4) 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-Cytc4 complexation involving nearly the same stabilization energy ∼ −15 × 103 kJ/mol, one through N-terminal Asp 15 and another -C terminal Glu 121 of Cytc4 with the Cu-bonded His 143 of RCy. The Asp 15:His 143 associated complex (DH) of Cytc4-RCy was stabilized by the intermolecular H-bonds of the carboxyl oxygen atoms Oδ1 and Oδ2 of Asp 15 with the Nϵ-atom of His 143 and Ob atoms of Ala 8 and Asp 5 (of Cytc4) with the Thr 146 and Phe 51 (of RCy). But the other Glu 121:His 143 associated complex (EH) of Cytc4-RCy was stabilized by the H-bonding interaction of the oxygen atoms Oϵ1 and Oϵ2 of Glu 121 with the Nϵ and Oγ 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 (Cytc4) 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 Cytc4) 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 Cytc4) 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 Cytc4.