Spectroscopic and Electrochemical Studies of CuA Protein from Thermus thermophilus and its Mutants

Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valence delocalized [Cu+1.5-Cu+1.5] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by confining the protein on glassy carbon electrode surface in presence of various surfactants. The irreversible denaturation of the protein on the electrode surface was overcome by using surfactant as promoter. The electrostatic and hydrophobic interactions probably play an important role in this process. The electrode thereby becomes gradually more accessible for electron transfer in presence of surfactants. A good promoter therefore not only forms a flexible bridge that compensates the electrostatic repulsion, but must also protect the protein from the hydrophobic patches on the electrode. The dependence of the redox potentials of CuA on pH, measured with electrochemistry showed the existence of different conformational change of CuA. Neomycin sulphate apparently has a function as promoter for CuA protein in this pH dependent electrochemical study. The redox potential as well as thermal stability of a protein is always influenced pH of the solution, the active site environment and also by the charges of the peptide. The mutagenic studies of CuA protein showed that the redox potential of CuA vary with the mutation in the loop region, which exerts subtle changes in the active site of the CuA. The thermal stability also decreases with the mutation in the loop region of CuA protein. Simultaneously spectroscopic and electrochemical measurements of the mutants replacing the bridging cysteines in the active core of the CuA showed that a mononuclear blue copper protein can be engineered inside the dinuclear CuA protein scaffold.