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

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/H2(18)O/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 H2(18)O sensitive bands at 3640 and 3634 cm(-1) shows the existence of weakly H-bonded water molecules.