FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase.
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 D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) 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 a(3) 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 pK(a) of about 8.3, but that remains protonated over the entire pH* range 6-9.3 in FR-CO. The pK(a) of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a(3) in MV oxidases, the extent of electron transfer from heme a(3) 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 a(3) 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 a(3) 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.
McMahon
Benjamin H
BH
Chemistry Division, Bioscience Division, and Center for Nonlinear Studies, Los Alamos National Laboratory, Michelson Res., Bioscience Division, Los Alamos, NM 87545, USA.
Fabian
Marian
M
Tomson
Farol
F
Causgrove
Timothy P
TP
Bailey
James A
JA
Rein
Francisca N
FN
Dyer
R Brian
RB
Palmer
Graham
G
Gennis
Robert B
RB
Woodruff
William H
WH
D000818:Animals; D002417:Cattle; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D018892:Proton-Motive Force; D017550:Spectroscopy, Fourier Transform Infrared
D006418:Heme; D003576:Electron Transport Complex IV