Detection of functional hydrogen-bonded water molecules with protonated/deprotonated key carboxyl side chains in the respiratory enzyme ba3-oxidoreductase

Abstract

The protonation/deprotonation of active carboxyl side chains by water networks forming the proton loading and exit sites in proteins are important steps in protein catalysis. An excellent system to study such basic principles is the heme–copper ba₃ from T. thermophilus because it utilizes one proton input channel and it delivers protons to the active site for both O₂ chemistry and proton pumping. We report the interaction of the heme a₃ Fe propionate-A and the Asp372–His376 pair which forms the valve for the exit pathway for the protons with internal water molecules in ba₃ oxidoreductase by light minus dark FTIR spectroscopy in conjunction with H₂O/H₂¹⁸O/D₂O exchange. The proton loading site consists of several water molecules including w941/w946 which are H-bonded to propionate-A–H⁺, acting as the Zundel cation. The detection of two H₂¹⁸O sensitive bands at 3640 and 3634 cm⁻¹ shows the existence of weakly H-bonded water molecules.