Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase

Abstract

Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R2,Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed.

A tyrosine residue deprotonates during oxygen reduction by the caa3 reductase from Rhodothermus marinus

“…This difference spectrum is very similar to that of the R. sphaeroides enzyme (green spectrum in Fig. 4B and [5]) as well as that of the P. denitrificans enzyme [13]. Prominent vibrational bands at 1548 and 1516 cm À1 are observed for the R. marinus oxygen reductase (red spectrum).…”

“…1B). Indeed, the P M À O difference spectrum of the latter exhibits a negative band at 1745 cm À1 that has previously been assigned to the C‚O stretching vibration of E286 [5] (Fig. 4A, green spectrum).…”

“…4A (red spectrum). The spectrum strongly resembles the P M À O difference spectrum for the aa3 oxygen reductase from R. sphaeroides [5], shown in amino acid tyrosine and the corresponding base, tyrosinate (Fig. 4A, black spectrum), the negative band at 1516 cm À1 is assigned to the phenyl ring stretching mode of tyrosine that downshifts to 1498 cm À1 (positive) upon formation of the tyrosinate.…”

“…We have shown by Fourier transform infrared (FTIR) spectroscopy that E286 acts as a proton shuttle in the oxygen reductase of R. sphaeroides because it is consecutively deprotonated and reprotonated twice within one catalytic turnover [5]. This finding has been challenged by FTIR experiments on the homologous enzyme from Paracoccus denitrificans [6] where only a change in H-bonding of the corresponding residue E278 has been reported for the P state.…”

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

“…Recent infrared experiments are elucidating the involvement of particular residues in the catalytic reaction sequence. 35,36 …”