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/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.

ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927

“…Based on the crystal structure cytochrome ba 3 from Thermus thermophilus contains a homodinuclear copper center (Cu A ), a low-spin heme b , and a heme a 3 -Cu B center [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. Cytochrome ba 3 catalyzes the reductions of oxygen (O 2 ) to water (H 2 O) and of nitric oxide (NO) to nitrous oxide (N 2 O), as well and the oxidation of carbon monoxide (CO) to carbon dioxide (CO 2 ) [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The photolyzed ba 3 -CO species is an excellent model for time-resolved spectroscopic studies [7,8,9,13,15,18,24].…”

“…Cytochrome ba 3 catalyzes the reductions of oxygen (O 2 ) to water (H 2 O) and of nitric oxide (NO) to nitrous oxide (N 2 O), as well and the oxidation of carbon monoxide (CO) to carbon dioxide (CO 2 ) [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The photolyzed ba 3 -CO species is an excellent model for time-resolved spectroscopic studies [7,8,9,13,15,18,24]. In the past, we used time-resolved Raman and step-scan FTIR (TRS 2 -FTIR) spectroscopy to probe the binding of CO to Cu B and the structural changes of the ring-A propionate of heme a 3 and D372 [7,8,9,10].…”

“…It was concluded that the trans / cis isomerization of the ring-D propionate plays a crucial role in controlling the orientations of “docked” CO between the heme rings-A and -D propionates and that the protein environment removes the barrier to the two orientations of CO [10]. The role of the heme a 3 -D372-H 2 O site and of ring-A propionate as proton carriers to the H 2 O pool, which is conserved among all structurally-known heme-copper oxidases, were reported [11,16,18,23]. The observation of deprotonated and protonated forms of heme a 3 rings-A and -D propionate and D372 indicated a protonic connectivity between the ring-A propionate, a H 2 O molecule and D372.…”

“…The observation of deprotonated and protonated forms of heme a 3 rings-A and -D propionate and D372 indicated a protonic connectivity between the ring-A propionate, a H 2 O molecule and D372. It was proposed that the environment of the ring-A heme a 3 propionate-D372-H 2 O moiety can contribute to proton motion [18,23]. …”

Fluorescence Responses of the Protonation and Deprotonation Processes between Phenolate and Phenol within Rosamine

“…[5] In addition to pH detection, the researches of protonated and deprotonated processes give a chance to view the structure-property relationship for functional dyes deeply. [6,7] Hydrogen bond plays an important role not only in the life process [8][9][10] but also in the construction of molecular systems. [11][12][13] Studies have shown that the formation of hydrogen bonds can inhibit the rotation between molecules, which can effectively immobilize the molecular structure, [14] and not only have an impact on the material melting point, boiling point, solubility, viscosity and other properties, [15] but also contribute to the nonradiative energy transitions of the excited state molecules, thus increasing the radiation transition.…”