Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

Abstract

The redox reactions of the cytochrome c oxidase from Puracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UVNIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surfacemodified electrode for all cofactors as indicated by the optical signals in the 400-700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reducedminus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm-' range reveal highly structured band features with major contributions in the amide 1(1620-1680 cm-') and amide II (1580-1520 cm-') range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm-', a positive peak in the reduced state (1733 cm-') and negative peak in the oxidized state (1745 cm-') are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Glu side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

The Protonation State of the Cross-linked Tyrosine during the Catalytic Cycle of Cytochrome c Oxidase

“…They also demonstrate a decrease in the amplitude of the 1308-cm Ϫ1 band at acidic conditions, indicating partial reprotonation of the tyrosine in the O state. The spectra of the R3 O transition measured with wild type enzyme at equilibrium conditions (25,36,37) showed high equivalence to the kinetic R3 O spectra of both mutated and wild type enzymes reported here.…”

A cytochrome c oxidase proton pumping mechanism that excludes the O2 reduction site

“…However, the direction of hydrogen bond structure can often provide a clear indication of protonation state changes of acidic amino acid residues. FTIR spectroscopy is a powerful tool for examination of the protonation/deprotonation state change, since the antisymmetric vibration of a protonated carboxyl group gives rise to a band near 1740 cm 31 whereas the analogous band of a deprotonated carboxyl group is found near 1580 cm 31 [21,22]. The FTIR di¡erence spectra of bovine heart cytochrome c oxidase in the oxidized state vs. the reduced state exhibit a positive peak at 1738 cm 31 and a negative peak at 1580 cm 31 [22].…”

Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

“…Application of spectro-electrochemical cells (47)(48)(49)(50)(51)(52), attenuated total reflection FTIR apparatus (41,42,(53)(54)(55)(56), and a photochemical reduction technique (57-60) enabled us to examine protein structural changes upon reduction of the redox metal centers of respiratory terminal oxidases. Alternatively, photodissociation of CO from the reduced enzyme revealed the local structural change surrounding the heme-copper binuclear center of the E. coli cytochrome bo (61,62).…”