TY - JOUR
T1 - FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase
AU - McMahon, Benjamin H.
AU - Fabian, Marian
AU - Tomson, Farol
AU - Causgrove, Timothy P.
AU - Bailey, James A.
AU - Rein, Francisca N.
AU - Dyer, R. Brian
AU - Palmer, Graham
AU - Gennis, Robert B.
AU - Woodruff, William H.
N1 - Funding Information:
Supported by grants from the National Institutes of Health (DK36263 to WHW, HL 16101 to RBG, and GM 55807 to GP), Welch Foundation Award C636 to GP, and a LANL Director's Postdoctoral Fellowship to BHM.
PY - 2004/4/12
Y1 - 2004/4/12
N2 - FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D2O (pH 6-9.3) between the MV-CO adduct (heme a3 and CuB reduced; heme a and Cu A oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a3 oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a 3 decreases from 1965.3 cm-1 at pH* ≤7 to 1963.7 cm-1 at pH* 9.3. In the CO adduct of the fully reduced enzyme (FR-CO), the CO stretching frequency is observed at 1963.46±0.05 cm -1, independent of pH. This indicates that in MV-CO there is a group proximal to heme a that deprotonates with a pKa of about 8.3, but that remains protonated over the entire pH* range 6-9.3 in FR-CO. The pKa of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a3 in MV oxidases, the extent of electron transfer from heme a3 to heme a shows a pH-dependent phase between pH 7 and 9, and a pH-independent phase at all pH's. The FTIR difference spectrum resulting from photolysis of MV-CO exhibits vibrational features of the protein backbone and side chains associated with (1) the loss of CO by the a3 heme in the absence of electron transfer, (2) the pH-independent phase of the electron transfer, and (3) the pH-dependent phase of the electron transfer. Many infrared features change intensity or frequency during both electron transfer phases and thus appear as positive or negative features in the difference spectra. In particular, a negative band at 1735 cm-1 and a positive band at 1412 cm-1 are consistent with the deprotonation of the acidic residue E242. Positive features at 1552 and 1661 cm-1 are due to amide backbone modes. Other positive and negative features between 1600 and 1700 cm-1 are consistent with redox-induced shifts in heme formyl vibrations, and the redox-linked protonation of an arginine residue, accompanying electron transfer from heme a3 to heme a. An arginine could be the residue responsible for the pH-dependent shift in the carbonyl frequency of MV-CO. Specific possibilities as to the functional significance of these observations are discussed.
AB - FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D2O (pH 6-9.3) between the MV-CO adduct (heme a3 and CuB reduced; heme a and Cu A oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a3 oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a 3 decreases from 1965.3 cm-1 at pH* ≤7 to 1963.7 cm-1 at pH* 9.3. In the CO adduct of the fully reduced enzyme (FR-CO), the CO stretching frequency is observed at 1963.46±0.05 cm -1, independent of pH. This indicates that in MV-CO there is a group proximal to heme a that deprotonates with a pKa of about 8.3, but that remains protonated over the entire pH* range 6-9.3 in FR-CO. The pKa of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a3 in MV oxidases, the extent of electron transfer from heme a3 to heme a shows a pH-dependent phase between pH 7 and 9, and a pH-independent phase at all pH's. The FTIR difference spectrum resulting from photolysis of MV-CO exhibits vibrational features of the protein backbone and side chains associated with (1) the loss of CO by the a3 heme in the absence of electron transfer, (2) the pH-independent phase of the electron transfer, and (3) the pH-dependent phase of the electron transfer. Many infrared features change intensity or frequency during both electron transfer phases and thus appear as positive or negative features in the difference spectra. In particular, a negative band at 1735 cm-1 and a positive band at 1412 cm-1 are consistent with the deprotonation of the acidic residue E242. Positive features at 1552 and 1661 cm-1 are due to amide backbone modes. Other positive and negative features between 1600 and 1700 cm-1 are consistent with redox-induced shifts in heme formyl vibrations, and the redox-linked protonation of an arginine residue, accompanying electron transfer from heme a3 to heme a. An arginine could be the residue responsible for the pH-dependent shift in the carbonyl frequency of MV-CO. Specific possibilities as to the functional significance of these observations are discussed.
KW - CcO
KW - Cytochrome c oxidase
KW - Cytochrome oxidase
KW - FR
KW - FTIR
KW - Fully reduced cytochrome c oxidase
KW - Glutamate
KW - MV
KW - MV-CO and FR-CO
KW - Mixed valence (two-electron reduced) cytochrome c oxidase
KW - Mixed-valence
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U2 - 10.1016/j.bbabio.2004.01.007
DO - 10.1016/j.bbabio.2004.01.007
M3 - Review article
C2 - 15100047
AN - SCOPUS:11144354250
SN - 0005-2728
VL - 1655
SP - 321
EP - 331
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
IS - 1-3
ER -